r ¹⁸ Fl RADIOTRACERS AND THEIR USES

TECHNICAL FIELD

The present invention relates to [ ¹⁸ F] radiotracers, their synthesis and their use in medical imaging, and in particular in PET imaging. In particular, the present invention relates to imaging of sodium/iodide symporter-expressing cells, such as those in the thyroid and thyroid cancer and other cancers, and imaging cells engineered to express the

sodium/iodide symporter, for the purposes of imaging and monitoring of diseases related to sodium/iodide symporter-expressing cells and tracking of cells engineered to express the sodium/iodide symporter after therapeutic administration of such engineered cells.

BACKGROUND

The sodium/iodide symporter (NIS) is a plasma membrane glycoprotein that mediates the active transport of iodide in the thyroid and other NIS-expressing cells or tissues. As a result, NIS is a well-established target in thyroid diseases, such as thyroid cancer, autoimmune disease and congenital hyperthyroidism. NIS has also found application as a reporter gene, engineered into cells for the purpose of locating them in the body by imaging with radionuclides or for selectively killing them with therapeutic radionuclides such as iodine-131 .

Radioiodines, such as ¹³¹ l, ¹²³ l and ¹²⁴ l have been used in the diagnosis and monitoring of these diseases for many years. For example, single photon emission computed tomography (SPECT) using ¹³¹ 1 and ¹²³ l has been investigated. However, the half-lives of

gamma-emitting radioiodines are longer than those required for imaging and thus absorbed radiation doses are unnecessarily high. In addition, positron emission tomography (PET) using ¹²⁴ l has been investigated. Alternative NIS tracers, such as [ ^99m Tc]pertechnetate, have been investigated for SPECT imaging of the thyroid. Despite some success of SPECT tracers, current SPECT tracers have limitations, for example in detecting smaller metastases and low volume diseases. Positron emission tomography (PET) is generally regarded as a superior imaging technique to SPECT as it offers improved resolution, sensitivity and quantification of radiotracer accumulation compared with SPECT, and it is more easily combined with magnetic resonance imaging. PET imaging of NIS-expressing tissues can be performed using ¹²⁴ l, a positron emitter with a half-life of 4.18 days. However, the half life of ¹²⁴ l is longer than necessary for most applications and it has a positron emission rate of only 23% alongside several high-energy gamma photons leading to increased background noise, increased radiation dose and poor image quality.

Various anions, such as CI0 ₄ ^" and BF ₄ ^" are known to be transported by NIS. ¹⁸ F is a commonly used radionuclide for PET. ¹⁸ F is available from most cyclotrons, and has a half-life of 1 10 minutes, which is appropriate for short-term imaging. Recently the present inventors described the synthesis and biological evaluation of [ ¹⁸ F]tetrafluoroborate - [ ¹⁸ F]BF ₄ - (Blower et al., Eur J Nucl Med Mol Imaging (2010) 37:2108-21 16). Unlabelled BF ₄ ^" was labelled with ¹⁸ F to produce [ ¹⁸ F]BF ₄ ^" by isotope exchange method under acidic conditions at 120 °C for 10 minutes. The procedure was simple, with straightforward labelling, purification and quality control, and the BF ₄ ^" starting material is inexpensive and widely available commercially. Thyroid uptake of [ ¹⁸ F]BF ₄ ^" was reported, but the reported specific activity was relatively low at around 1 GBq/μιτιοΙ. This leads to a risk that imaging may be compromised by saturation of NIS in vivo if high administered activities are needed to ensure optimal image quality. A method was recently reported by the present inventors to label [ ¹⁸ F]BF ₄ ^" with higher specific activity but even with the new method the specific activity is lower than would be desirable for some applications (Blower et al., Eur J Nucl Med Mol Imaging Res 2016).

Accordingly, there is still a need for effective ¹⁸ F-labelled NIS imaging agents.

SUMMARY OF THE INVENTION

The present inventors have sought to provide ¹⁸ F-labelled NIS imaging agents with suitable properties for use in PET imaging, in particular for PET imaging of the thyroid or NIS expressing cells in a subject. The present inventors have achieved this by synthesising new radiotracers including [ ¹⁸ F]-labelled fluorosulfate [ ¹⁸ F]-labelled hexafluorophosphate or

[ ¹⁸ F]-labelled diflurophosphate (also referred to as difluoromonophosphate) anions.

In a first aspect, the present invention provides a radiotracer compound comprising

[ ¹⁸ F]S03F ^" , [ ¹⁸ F]PF6 ^" or [ ¹⁸ F]P02F2 ^" . Such radiotracer compounds may be produced in a composition with high specific activity and high radiochemical purity. Such radiotracer compounds may also have a high and NIS-specific uptake in NIS-expressing cells in vitro and excellent specific uptake of the tracer in NIS-expressing tissues (namely, the thyroid, stomach and salivary glands) in animal model in vivo studies. In a second aspect, the present invention provides a radiotracer composition comprising a radiotracer compound according to the first aspect.

In a third aspect, the present invention provides a method of synthesising a radiotracer compound comprising [ ¹⁸ F]SOsF ^" , [ ¹⁸ F]PF6 ^" or [ ¹⁸ F]P02F2 ^" , the method comprising the step of (i) reacting:

(a) a non-radioactive precursor capable of reacting with [ ¹⁸ F]-fluoride to produce the radiotracer compound of the first aspect; with

(b) [ ¹⁸ F]-fluoride. Typically, the non-radioactive precursor capable of reacting with [ ¹⁸ F]-fluoride ([ ¹⁸ F]F ^" ) to produce the radiotracer compound comprising [ ¹⁸ F]SOsF ^" is SO3 or an adduct of SO3.

Typically, the non-radioactive precursor capable of reacting with [ ¹⁸ F]-fluoride ([ ¹⁸ F]F ^" ) to produce the radiotracer compound comprising is PF ₅ , PCI5 or an adduct of PF ₅ .

Typically, the non-radioactive precursor capable of reacting with [ ¹⁸ F]-fluoride ([ ¹⁸ F]F ^" ) to produce the radiotracer compound comprising [ ¹⁸ F]PF6 ^" is P4O10, PF ₅ , PCI5 or an adduct of

Using an adduct of a Lewis base and SO3, PF ₅ , PO2F or P2O5 as the reactant (a) in the above synthetic method of the third aspect may provide the radiotracer compound of the first aspect by a simple synthesis with a high specific activity.

In a fourth aspect, the present invention provides a kit for use in the production of a radiotracer compound of the first aspect, wherein the kit comprises two or more of the following components:

(i) a unit containing a non-radioactive precursor capable of reacting with

[ ¹⁸ F]-fluoride to produce the radiotracer compound of the first aspect;

(ii) a unit containing a fluoride-extracting solid material for extracting fluoride from a solution;

(iii) a unit containing one of: (a) a fluorosulfate-extracting solid material for

extracting fluorosulfate from a solution, (b) a hexafluorophosphate-extracting solid material for extracting hexafluorophosphate from a solution, or (c) a difluoromonophosphate-extracting solid material for extracting difluoromonophosphate from a solution; and

(iv) instructions for use of the kit in the production of a radiotracer compound of the first aspect. In some embodiments of the fourth aspect, the components of the kit are housed in a cassette adapted to be used with an automated radiotracer synthesizer apparatus.

In a fifth aspect, the present invention provides a radiotracer compound according to the first aspect or a radiotracer composition according to the second aspect for use in a method of medical imaging.

In a sixth aspect, the present invention provides a method of medical imaging a subject using a radiotracer compound according to the first aspect or a radiotracer compound composition according to the second aspect, the method including the steps of:

(i) administering an effective amount of the radiotracer compound to the subject; and then

(ii) medically imaging the subject, preferably PET imaging the subject. The subject of the methods above is typically a human subject or an animal subject in experimental imaging.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will be described, in particular with reference to the Examples and drawings.

Brief Description of Drawings

Figure 1 a shows a graph of uptake of ^99m Tc04 ^" in a human NIS (hNIS) expressing cell line (HCT1 16-C19) in the presence KSO3F at various concentrations to determine the IC50 value

Figure 1 b shows a graph comparing uptake of ^99m Tc04- in a cell line expressing a

hNIS/TagRFP fusion protein, MTLn3E.D34-CXCR4-GFP.hNIS-TagRFP (also known as 3E.A-hNIS, Fruhwirth et al., 2014), in the presence of various concentrations of SO3F ^" and BF ₄ ^" to determine their IC50. Concentration units (x axis) are 10 ^x M.

Figure 2a shows uptake of [ ¹⁸ F]S0 ₃ F- in HCT1 16-C19 (hNIS-expressing) and HCT1 16 (non- hNIS-expressing) cells, with and without perchlorate blocking (left); Figure 2b shows uptake of [ ¹⁸ F]S0 ₃ P over time in HCT1 16-C19 cells (right).

Figure 2c shows uptake of [ ¹⁸ F]SOsF ^" and ^99m Tc0 ₄ ^" simultaneously incubated with

MTLn3E.D34-CXCR4-GFP.hNIS-TagRFP cells (also known as 3E.A-hNIS cells, Fruhwirth et al., 2014), and likewise [ ¹⁸ F]BF ₄ ^" and 99mTc04- simultaneously incubated with 3E.A-hNIS cells. Uptake of [ ¹⁸ F]S0 ₃ F ^" and ^99m Tc0 ₄ ^" are both reduced when in the presence of [ ¹⁸ F]BF ₄ ^" , showing the effect of the low specific activity of [ ¹⁸ F]BF ₄ ^" .

Figure 3 shows maximum intensity projection PET/CT images of normal mice injected with [ ¹⁸ F]SC>3F ^" , at a 30 minute period commencing at the time of [ ¹⁸ F]SOsF ^" injection (Figure 3a), and at a 30 minute period commencing at the time of [ ¹⁸ F]SOsF ^" injection and with NaCI0 ₄ administration one hour before SO3F administration (250 mg/kg) (Figure 3b)..

Figure 4 shows ex vivo biodistribution of [ ¹⁸ F]SOsF ^" at 2 hours with and without NaCI0 ₄ (250 mg/kg) administration expressed as standardised uptake values (n = 3). Error bars represent SEM.

Figure 5 shows in vivo uptake of [ ¹⁸ F]PF ₆ ^" in HCT1 16-C19 (hNIS-expressing) and HCT1 16 (non-hNIS-expressing) cells, with and without perchlorate blocking.

Radiotracer Compounds

The present invention provides a radiotracer compound including the radiolabeled anion [ ¹⁸ F]SC>3F ^" , [ ¹⁸ F]PF6 ^" or [ ¹⁸ F]PC>2F2 ^" . In one embodiment, the present invention provides a radiotracer compound including the radiolabeled anion In another embodiment, the present invention provides a radiotracer compound including the radiolabeled anion [ ¹⁸ F]PF6 ^" . In a further embodiment, the present invention provides a radiotracer compound including the radiolabeled anion [ ¹⁸ F]P02F2 ^" . The compound will typically include a cation as a counter-ion. The cation may be a monovalent cation or a multivalent cation. In some embodiments, the cation is a pharmaceutically acceptable cation. Examples of

pharmaceutically acceptable salts (and therefore cations) are discussed in Berge et al., 1977, "Pharmaceutically Acceptable Salts," J. Pharm. Sci., Vol. 66, pp. 1 -19. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Na ⁺ and K ⁺ , alkaline earth cations such as Ca ²⁺ and Mg ²⁺ , and other cations such as Al ³⁺ . Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e., NH4 ⁺ ) and substituted ammonium ions (e.g., NH3R ⁺ , NhbF^, NHR3 ⁺ , NR ₄ ⁺ ). Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine,

diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH3) ₄ ⁺ . A further example of suitable organic cations include pyridinium and pyridinium derivatives.

If the compound includes a multivalent (e.g., divalent, trivalent, tetravalent) cation, the radiotracer compound may include one or more spectator anions (in addition to [ ¹⁸ F]S03F ^" , [ ¹⁸ F]PF6 ^" or [ ¹⁸ F]PC>2F2 ^" )- Examples of suitable inorganic spectator anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric, nitrous, phosphoric, and phosphorous.

Examples of suitable organic spectator anions include, but are not limited to, those derived from the following organic acids: 2-acetyoxybenzoic, acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric, edetic, ethanedisulfonic, ethanesulfonic, fumaric, glucheptonic, gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalene carboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic, methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic, phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic, succinic, sulfanilic, tartaric, toluenesulfonic, and valeric. Examples of suitable polymeric organic spectator anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethyl cellulose.

In some embodiments, the radiotracer compound is represented by the formula [ ¹⁸ F]XS03F, [ ¹⁸ F]XPF6 or [ ¹⁸ F]XPC>2F2 where X is a monovalent cation. In one embodiment, X is a pharmaceutically acceptable monovalent cation. In one embodiment, X is selected from the group consisting of Na ⁺ , K ⁺ , ammonium ion (i.e., NH ₄ ⁺ ), substituted ammonium ions (e.g., NH ₃ R ⁺ , NH ₂ R ₂ ⁺ , NHR ₃ ⁺ , NR ₄ ⁺ ) and pyridinium (C ₅ H ₆ N ⁺ ). Method of Synthesising Radiotracer Compounds and Radiotracer Compound Compositions The present invention also provides methods of synthesising radiotracer compounds described herein. At its most general, the present invention provides a method of synthesising a compound comprising [ ¹⁸ F]SOsF ^" , [ ¹⁸ F]PF6 ^" or [ ¹⁸ F]PC>2F2 ^" , the method comprising the step of (i) reacting:

(a) a non-radioactive precursor capable of reacting with [ ¹⁸ F]-fluoride to produce the radiotracer compound described herein; with

(b) [ ¹⁸ F]-fluoride.

In one embodiment, the method synthesises a radiotracer compound including the radiolabeled anion In another embodiment, the method synthesises a radiotracer compound including the radiolabeled anion [ ¹⁸ F]PF6 ^" . In a further embodiment, the method synthesises a radiotracer compound including the radiolabeled anion

[ ¹⁸ F]P0 ₂ F ₂ \ The generation of [ ¹⁸ F]-fluoride ([ ¹⁸ F]F ^" ) is well known. For example, [ ¹⁸ F]-fluoride may be generated from ¹⁸ 0-water using a cyclotron. In some embodiments, the non-radioactive precursor capable of reacting with [ ¹⁸ F]-fluoride to produce the radiotracer compound comprising [ ¹⁸ F]SOsF ^" may be SO3 gas. SO3 gas may be used as reactant (a) to yield [ ¹⁸ F]SOsF ^" . The gas may be reacted with a source of

[ ¹⁸ F]-fluoride using known techniques. For example, SO3 gas can be bubbled through a solution (aqueous or non-aqueous) containing [ ¹⁸ F]-fluoride. Alternatively, SO3 gas may be condensed on chilled dry [ ¹⁸ F]-fluoride on an inert surface, such as glass. As a further alternative, SO3 gas may be passed through an anion exchange column loaded with

[ ¹⁸ F]-fluoride.

Alternatively, the non-radioactive precursor capable of reacting with [ ¹⁸ F]-fluoride to produce the radiotracer compound comprising [ ¹⁸ F]SOsF ^" (reactant (a)) may be an adduct of SO3, particularly, a Lewis adduct formed between a Lewis base and SO3. The present inventors have found that using a Lewis adduct of a Lewis base and SO3 as reactant (a) provides samples of the radiotracer compound comprising [ ¹⁸ F]SC>3F ^" with a high specific activity. In some embodiments, the non-radioactive precursor capable of reacting with [ ¹⁸ F]-fluoride to produce the radiotracer compound comprising [ ¹⁸ F]PF6 ^" may be PF ₅ gas. The gas may be reacted with a source of [ ¹⁸ F]F ^" using known techniques. For example, PF ₅ gas can be bubbled through a solution (aqueous or non-aqueous) containing [ ¹⁸ F]F\ Alternatively, PF ₅ gas may be condensed on chilled dry [ ¹⁸ F]F ^" on an inert surface, such as glass. As a further alternative, PF ₅ gas may be passed through an anion exchange column loaded with [ ¹⁸ F]F\

In alternative embodiments, the non-radioactive precursor capable of reacting with [ ¹⁸ F]- fluoride to produce the radiotracer compound comprising [ ¹⁸ F]PF6 ^" may be PCI5. Typically, a mixture of [ ¹⁸ F]P02F2 ^" and [ ¹⁸ F]PF6 ^" is generated when PCI5 and PF ₅ are used. The [ ¹⁸ F]PF6 ^" may be separated, for example by hydrolysis under basic conditions. Alternatively, the non-radioactive precursor capable of reacting with [ ¹⁸ F]-fluoride to produce the radiotracer compound comprising [ ¹⁸ F]PF6 ^" (reactant (a)) may be an adduct of PF ₅ , in particular, a Lewis adduct formed between a Lewis base and PF ₅ . An adduct of a Lewis base and PF ₅ as the reactant (a) in the above synthetic method of the third aspect may provide the radiotracer compound comprising [ ¹⁸ F]PF6 ^" of the first aspect by a simple synthesis.

In some embodiments, the non-radioactive precursor capable of reacting with [ ¹⁸ F]-fluoride to produce the radiotracer compound comprising [ ¹⁸ F]PC>2F2 ^" may be PF ₅ gas. The gas may be reacted with a source of [ ¹⁸ F]F ^" using known techniques. For example, PF ₅ gas can be bubbled through a solution (aqueous or non-aqueous) containing [ ¹⁸ F]F\ Alternatively, PF ₅ gas may be condensed on chilled dry [ ¹⁸ F]F ^" on an inert surface, such as glass. As a further alternative, PF ₅ gas may be passed through an anion exchange column loaded with [ ¹⁸ F]F\

In alternative embodiments, the non-radioactive precursor capable of reacting with

[ ¹⁸ F]-fluoride to produce the radiotracer compound comprising [ ¹⁸ F]PC>2F2 ^" may be PCI5.

Typically, a mixture of [ ¹⁸ F]P02F2 ^" and [ ¹⁸ F]PF6 ^" is generated when PCI5 and PF ₅ are used. The [ ¹⁸ F]PC>2F2 ^" may be separated using suitable techniques. Alternatively, the [ ¹⁸ F]P02F2 ^" and [ ¹⁸ F]PF6 ^" are not separated from the reaction mixture. In other words, the radiotracer composition as described herein may include a compound comprising [ ¹⁸ F]P02F2 ^" and a compound comprising [ ¹⁸ F]PF6 ^" . In some embodiments, P2O5 (as P4O10) may be used as reactant (a) to yield [ ¹⁸ F]P02F2 ^" . P2O5 (as P4O10) may be reacted with a source of [ ¹⁸ F]F ^" using known techniques.

Alternatively, the reactant (a) is a Lewis adduct formed between a Lewis base and PO2F. An adduct of a Lewis base and PO2F2 as the reactant (a) in the above synthetic method of the third aspect may provide the radiotracer compound comprising [ ¹⁸ F]P02F2 ^" of the first aspect by a simple synthesis.

As described herein, adduct and Lewis adduct take their conventional meanings. In other words, an adduct is a species (AB) formed from two individual species (A and B) without loss of atoms from the individual species. A Lewis adduct (also known as a Lewis acid/base adduct) is an adduct formed between a Lewis acid (SO3 for this invention) and a Lewis base.

The Lewis base may be any Lewis base, and Lewis bases are known per se. Examples of

Lewis bases include, but are not limited to:

amines, such as ammonia, alkyl amines (e.g. NH3-xR ¹ x, where Ri is an alkyl group or an aryl group and x is an integer selected from the group consisting of 1 , 2 or 3), pyridine, and pyridine derivatives;

phosphines (e.g.PR ² 3- _y R ³ _y , where R ² is an alkyl group, R ³ is an aryl group and y is an integer selected from the group consisting of 0, 1 , 2 or 3);

compounds containing O or S in oxidation state 2, such as water, ethers and ketones;

simple anions such as fluoride, chloride and sulfate; and

electron-rich ττ-system Lewis bases, such as ethyne, ethane and benzene.

Specific examples of Lewis bases include trimethylamine (ΜββΝ), triethylamine (ΕίβΝ), quinuclidine, pyridine, acetonitrile, diethyl ether (Et.20), tetrahydrofuran (THF), 1 ,4-dioxane, acetone, ethyl acetate (EtOAc), dimethylacetamide (DMA), dimethylsulfoxide (DMSO), tetrahydrothiophene, fluoride, chloride, sulfate and trimethylphosphine.

In some embodiments, the Lewis adduct of a Lewis base and SO3 in (a) is soluble in an organic solvent. In particular, the Lewis base in (a) may be pyridine. In other words, the reactant in (a) may be a sulfur trioxide-pyridine complex.

In some embodiments, the Lewis adduct of a Lewis base and PF ₅ in (a) is soluble in an organic solvent. In particular, the Lewis base in (a) may be pyridine. In other words, the reactant in (a) may be a phosphorus pentafluoride-pyridine complex.

In some embodiments, the Lewis adduct of a Lewis base and PO2F in (a) is soluble in an organic solvent. In particular, the Lewis base in (a) may be pyridine. In other words, the reactant in (a) may be a monofluoromonophosphate-pyridine complex.

In some embodiments, the reaction in step (i) includes water, an organic solvent or a mixture thereof. In one embodiment the reaction in step (i) includes water, acetonitrile, or a mixture thereof. In some embodiments, at least part of the reaction in step (i) is performed at 30 °C or more, 50 °C or more, 70 °C or more, or 75 °C or more. In other embodiments, the reaction in step (i) is performed at temperatures no greater than 100 °C, no greater than 90 °C, or no greater than 85 °C. In one embodiment, at least part of the reaction is performed in the range of 50 to 100 °C, in the range of 70 to 90 °C, or in the range of 75 to 85 °C.

In some embodiments, the reaction time in step (i) is 30 minutes or less, 20 minutes or less or 15 minutes or less. In some embodiments, the reaction time in step (i) is 5 minutes or more. In one embodiment, the reaction time in step (i) is in the range of 5 to 15 minutes. In some embodiments, the reaction in step (i) includes a Br0nsted base. Bransted bases are known per se. In particular embodiments, the Bransted base is KHCO3 or K2CO3. In some embodiments, the reaction in step (i) includes a cryptand. The cryptand is typically included as a complex with the counter-ion of [ ¹⁸ F]F\ In one embodiment, the cryptand is Kryptofix K2.2.2 (K222). In some embodiments, reactant [ ¹⁸ F]F ^" in (b) is provided as [ ¹⁸ F]NaF or [ ¹⁸ F]KF. In some embodiments, reactant [ ¹⁸ F]F ^" in (b) is provided as a cryptand complex of [ ¹⁸ F]NaF or [ ¹⁸ F]KF. In some embodiments, the method further includes a step of (ii) purification after reaction step (i) has been performed. Such a purification step may remove some or all chemical or radiochemical impurities from the reaction.

Purification step (ii) may include passing the reacted mixture of step (i) (i.e. the mixture after reaction) over one or more solid phase extraction units or devices. The purification step may include passing the reacted mixture of step (i) over a fluoride-extracting solid material and/or one of (a) a fluorosulfate-extracting solid material, (b) a hexafluorophosphate-extracting solid material or (c) a difluoromonophosphate-extracting solid material. The reacted mixture of step (i) may be diluted with a solvent before passing the reacted mixture over the solid phase extraction device. The dilution solvent may be water.

The fluoride-extracting solid material is any material capable of extracting fluoride (such as unreacted [ ¹⁸ F]F ^" ) from the reacted mixture of step (i). The fluoride-extracting solid material may be contained in a column or contained in a cartridge. The fluoride-extracting solid material may be an aluminium-containing mineral or a calcium-containing mineral. In some embodiments, the fluoride-extracting solid material is selected from the group consisting of alumina, aluminium hydroxide and hydroxyapatite. The choice of fluorosulfate-extracting solid material, hexafluorophosphate-extracting solid material and difluoromonophosphate-extracting solid material will depend on the radiotracer anion being synthesised. In other words, a fluorosulfate-extracting solid material may be used when synthesising [ ¹⁸ F]S03F ^" ; a hexafluorophosphate-extracting solid material may be used when synthesising [ ¹⁸ F]PF6 ^" ; and a difluoromonophosphate-extracting solid material may be used when synthesising or [ ¹⁸ F]P02F2 ^" .

Purification of r ¹⁸ FlSQ3p

Purification step (ii) may include passing the reacted mixture of step (i) (i.e. the mixture after reaction) over one or more solid phase extraction units or devices. The purification step may include passing the reacted mixture of step (i) over a fluoride-extracting solid material and/or a fluorosulfate-extracting solid material. The reacted mixture of step (i) may be diluted with a solvent before passing the reacted mixture over the solid phase extraction device. The dilution solvent may be water.

The fluorosulfate-extracting solid material may be any material capable of extracting fluorosulfate from the reacted mixture of step (i) (or the eluate of other solid phase extraction devices used on the reacted mixture of step (i)), such as an anion-exchanger solid material. The fluorosulfate anion-exchanger solid material may be a silica-based, hydrophilic, strong anion-exchanger (such as a SAX, strong anion exchanger with quaternary ammonium functional group, e.g. tetraalkylammonium-derivatised silica;) or a weak anion exchanger (such as a WAX, a weak anion exchanger with a tertiary amine functional group, or a primary amine derivatised silica). Such anion-exchangers are known and widely available. The pore size of the anion exchanger is not limited. In some embodiments, the pore size of the anion exchanger is in the range of 200 to 400 A. In some embodiments, the fluorosulfate anion exchanger solid material is the material contained in a QMA cartridge or a SAX cartridge (strong anion exchanger with quaternary ammonium functional group). In some embodiments, the purification step (ii) includes passing the reacted mixture of step (i) over both a fluoride-extracting solid material and a fluorosulfate-extracting solid material. The order in which the reacted mixture is passed over these solid phase extraction devices is not limited. In one embodiment, the reacted mixture of step (i) is diluted with a solvent, the diluted reacted mixture is passed over a fluoride-extracting solid material and then the eluate from the fluoride-extracting solid material is passed over a fluorosulfate-extracting solid material.

In further embodiments, step (ii) of purification includes passing the reacted mixture over a fluorosulfate-extracting solid material and further washing the fluorosulfate-extracting solid material with an eluting solution. [ ¹⁸ F]SC>3F ^" will generally be adsorbed by the

fluorosulfate-extracting solid material from the reacted mixture of step (i) and the eluting solution will displace [ ¹⁸ F]S03F ^" into the eluting solution to provide a radiotracer solution including The eluting solution may be a NaCI solution or a buffer solution. The buffer solution may be any solution with a sufficient ionic content to elute the radiotracer compound. Alternatively, if a weak anion exchanger (WAX or Nh ) is used, the eluting solution may be a basic solution. Such a basic solution increases the pH to cancel the anion binding properties of the column and elute the product. Purification of r ¹⁸ F1PF _R -

Purification step (ii) may include passing the reacted mixture of step (i) (i.e. the mixture after reaction) over one or more solid phase extraction devices. The purification step may include passing the reacted mixture of step (i) over a fluoride-extracting solid material and/or a hexafluorophosphate-extracting solid material. The reacted mixture of step (i) may be diluted with a solvent before passing the reacted mixture over the solid phase extraction device. The dilution solvent may be water. The hexafluorophosphate-extracting solid material may be any material capable of extracting hexafluorophosphate from the reacted mixture of step (i) (or the eluate of other solid phase extraction devices used on the reacted mixture of step (i)). The

hexafluorophosphate-extracting solid material may be a silica-based, hydrophilic, strong anion-exchanger (such as a SAX, strong anion exchanger with quaternary ammonium functional group, e.g. tetraalkylammonium-derivatised silica;) or a weak anion exchanger (such as a WAX, a weak anion exchanger with a tertiary amine functional group, or a primary amine derivatised silica). Such anion-exchangers are known and widely available. The pore size of the anion exchanger is not limited. In some embodiments, the pore size of the anion exchanger is in the range of 200 to 400 A. In some embodiments, the

hexafluorophosphate-extracting solid material is the material contained in a QMA cartridge or a SAX cartridge (strong anion exchanger with quaternary ammonium functional group).

In some embodiments, the purification step (ii) includes passing the reacted mixture of step (i) over both a fluoride-extracting solid material and a hexafluorophosphate-extracting solid material. The order in which the reacted mixture is passed over these solid phase extraction devices is not limited. In one embodiment, the reacted mixture of step (i) is diluted with a solvent, the diluted reacted mixture is passed over a fluoride-extracting solid material and then the eluate from the fluoride-extracting solid material is passed over a

hexafluorophosphate-extracting solid material.

In further embodiments, step (ii) of purification includes passing the reacted mixture over a hexafluorophsosphate-extracting solid material and further washing the

hexafluorophosphate-extracting solid material with an eluting solution. [ ¹⁸ F]PF6 ^" will generally be adsorbed by the hexafluorophsophate-extracting solid material from the reacted mixture of step (i) and the eluting solution will displace [ ¹⁸ F]PF6 ^" into the eluting solution to provide a radiotracer solution including [ ¹⁸ F]PF6 ^" . The eluting solution may be a NaCI solution or a buffer solution. The buffer solution may be any solution with a sufficient ionic content to elute the radiotracer compound. Alternatively, if a weak anion exchanger (WAX or Nh ) is used, the eluting solution may be a basic solution. Such a basic solution increases the pH to cancel the anion binding properties of the column and elute the product. Purification of i ¹⁸ FlPO _? F

Purification step (ii) may include passing the reacted mixture of step (i) (i.e. the mixture after reaction) over one or more solid phase extraction devices. The purification step may include passing the reacted mixture of step (i) over a fluoride-extracting solid material and/or a difluoromonophosphate-extracting solid material. The reacted mixture of step (i) may be diluted with a solvent before passing the reacted mixture over the solid phase extraction device. The dilution solvent may be water.

The difluoromonophosphate-extracting solid material may be any material capable of extracting difluoromonophosphate from the reacted mixture of step (i) (or the eluate of other solid phase extraction devices used on the reacted mixture of step (i)). The

difluoromonophosphate -extracting solid material may be a silica-based, hydrophilic, strong anion-exchanger (such as a SAX, strong anion exchanger with quaternary ammonium functional group, e.g. tetraalkylammonium-derivatised silica;) or a weak anion exchanger (such as a WAX, a weak anion exchanger with a tertiary amine functional group, or a primary amine derivatised silica). Such anion-exchangers are known and widely available. The pore size of the anion exchanger is not limited. In some embodiments, the pore size of the anion exchanger is in the range of 200 to 400 A. In some embodiments, the

difluoromonophosphate-extracting solid material is the material contained in a QMA cartridge or a SAX cartridge (strong anion exchanger with quaternary ammonium functional group).

In some embodiments, the purification step (ii) includes passing the reacted mixture of step (i) over both a fluoride-extracting solid material and a difluoromonophosphate-extracting solid material. The order in which the reacted mixture is passed over these solid phase extraction devices is not limited. In one embodiment, the reacted mixture of step (i) is diluted with a solvent, the diluted reacted mixture is passed over a fluoride-extracting solid material and then the eluate from the fluoride-extracting solid material is passed over a

difluoromonophosphate-extracting solid material.

In further embodiments, step (ii) of purification includes passing the reacted mixture over a difluoromonophosphate-extracting solid material and further washing the

difluoromonophosphate-extracting solid material with an eluting solution. [ ¹⁸ F]P02F2 ^" will generally be adsorbed by the difluoromonophosphate-extracting solid material from the reacted mixture of step (i) and the eluting solution will displace [ ¹⁸ F]P02F2 ^" into the eluting solution to provide a radiotracer solution including [ ¹⁸ F]P02F2 ^" . The eluting solution may be a NaCI solution or a buffer solution. The buffer solution may be any solution with a sufficient ionic content to elute the radiotracer compound. Alternatively, if a weak anion exchanger (WAX or NH2) is used, the eluting solution may be a basic solution. Such a basic solution increases the pH to cancel the anion binding properties of the column and elute the product.

Radiotracer Compositions

In another aspect, the present invention provides a radiotracer composition comprising a radiotracer compound including the radiolabeled anion [ ¹⁸ F]SOsF ^" , [ ¹⁸ F]PF6 ^" or [ ¹⁸ F]PC>2F2 ^" . In one embodiment, the present invention provides a radiotracer composition comprising a radiotracer compound including the radiolabeled anion In another embodiment, the present invention provides a radiotracer composition comprising a radiotracer compound including the radiolabeled anion [ ¹⁸ F]PF6 ^" . In a further embodiment, the present invention provides a radiotracer composition comprising a radiotracer compound including the radiolabeled anion [ ¹⁸ F]P0 ₂ F ₂ ^" .

In some embodiments, the radiotracer composition further includes a pharmaceutically acceptable carrier, excipient or biocompatible carrier. Compositions including a radiotracer compound described herein and a pharmaceutically acceptable carrier, excipient or biocompatible carrier may be in a form suitable for administration to a subject for diagnostic medical imaging. Alternatively, the compositions may be in a form suitable for administration to experimental animals for experimental in vivo imaging. Such compositions may also be referred to as radiopharmaceutical preparations.

Alternatively, the radiotracer compositions described herein may be a stock radiotracer composition. Such stock radiotracer compositions may be formulated further (e.g. removal of solvent; further purification; sterilisation; addition of one or more solvents, including dilution of solvent already present; addition of one or more pharmaceutically acceptable carriers, excipients or biocompatible carriers) to prepare a composition suitable for administration to a subject. In some embodiments, the stock radiotracer composition is a radiotracer solution (including [ ¹⁸ F]SC>3F ^" , [ ¹⁸ F]PF6 ^" or [ ¹⁸ F]PC>2F2 ^" ) eluted from a purification step (ii) from the method described above.

The radiotracer composition may include one or more components residual from the synthesis of the radiotracer compound described herein. In general, it is beneficial to produce the radiotracer composition in as short a time as possible to avoid loss of specific activity through radioactive decay. The radiotracer composition may include salts, such as NaCI, pyridinium salts, KHC0 ₃ , K ₂ C0 ₃ , NaHCOs, Na ₂ C0 ₃ , NH4HCO3, and (NH ₄ ) ₂ C0 ₃ , pyridine and cryptands, such as Kryptofix 2.2.2 (K222). Typically, these components, in total, form less than 20 wt.%, less than 15 wt.%, less than 10 wt.% or less than 5 wt.% of the radiotracer composition. In particular, the impurity level of pyridine may be 400 ppm or lower, 300 ppm or lower, or 200 ppm or lower per dose. The impurity level of the cryptand(s) may be 100 μg mL or lower, 75 μg mL or lower, or 50 μg mL or lower per dose. In some embodiments, the radiotracer compositions may include residual ¹⁸ F\ However, the levels of ¹⁸ F ^" in the radiotracer composition are typically very low (less than 0.001 wt.%). The radiotracer composition may have a radiochemical purity of 90 % or more. As used herein, radiochemical purity is the proportion of radioactivity in the composition attributed to the radiotracer compound. In other words, 90% or more of the radioactivity of the radiotracer composition may come from the radiotracer compound. The remaining radioactivity (if any) may come from unreacted or excess ¹⁸ F fluoride anions ( ¹⁸ F ^" ), or any other impurity. In some embodiments, the radiotracer composition has a radiochemical purity of 95 % or more, 98 % or more, or 99 % or more.

In some embodiments, the radiotracer composition has a specific activity of 1 GBq/μηΊθΙ or more, 10 GBq/μηΊθΙ or more, 30 GBq/μηΊθΙ or more, or 50 GBq/μηΊθΙ or more. Immediately after the radiolabelling of the radiotracer compound described herein, the specific activity may be higher. The radiotracer composition may have a specific activity of 10 GBq/μηΊθΙ or more, 30 GBq/μη-ιοΙ or more, 50 GBq/μη-ιοΙ or more, or 100 GBq/μη-ιοΙ as measured up to 15 minutes after radiolabelling of the radiotracer compound. The radiotracer composition may then be administered within hours of radiolabelling in order to achieve a satisfactory specific activity at administration.

Pharmaceutically acceptable carriers, excipients and biocompatible carriers are generally known. The pharmaceutically acceptable carrier, excipient or biocompatible carrier may be a fluid, e.g. a liquid, in which the radiotracer compound can be dissolved or suspended. Examples of pharmaceutically acceptable carriers, excipients or biocompatible carriers include, but are not limited to, sterile, pyrogen-free water (suitable for injection into a subject); an aqueous solution, such as saline (optionally balanced so that the final composition is isotonic or not hypotonic); sugars (such as glucose or sucrose); sugar alcohols (such as sorbitol or mannitol); buffers (such as phosphate buffered saline); an aqueous solution of one or more tonicity-adjusting substances (e.g. salts of plasma cations with biocompatible counter-ions); pharmaceutically acceptable solubilisers (such as cyclodextrins, and/or surfactants, e.g. Tween, Pluronic, and/or phospholipids); glycols (such as glycerol); other non-ionic polyol materials (such as polyethylene glycols or propylene glycols); vegetable oils; pharmaceutically acceptable antimicrobial preservatives (such as benzyl alcohol; phenol; cresol; a paraben, e.g. methyl paraben, ethyl paraben, propyl paraben, butyl paraben; thiomersla; and/or cetrimide); bacteriostats; suspending agents; thickening agents; solutes which render the formulation isotonic with the blood (or other relevant bodily fluid) of the intended recipient; and/or pharmaceutically acceptable stabilisers or antioxidants (such as para-aminobenzoic acid, gentisic acid or ascorbic acid). The pharmaceutically acceptable carrier, excipient or biocompatible carrier may additionally or alternatively include one or more biocompatible organic solvents at a pharmaceutically tolerable level, such as ethanol or acetonitrile. Examples of suitable isotonic carriers for use in such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection.

The term "pharmaceutically acceptable," as used herein, pertains to compounds, ingredients, materials, compositions, dosage forms, etc., which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of the subject in question (e.g., human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, diluent, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation.

Further suitable carriers, diluents, excipients, etc. can be found in standard pharmaceutical texts, for example, Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton, Pa., 1990; and Handbook of Pharmaceutical Excipients, 5th edition, 2005.

The formulations may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the radiotracer compound with a carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the compound with carriers (e.g., liquid carriers, finely divided solid carrier, etc.), and then shaping the product, if necessary.

When the radiotracer composition includes water (in other words, is an aqueous solution), the pH of the radiotracer composition may be a pharmaceutically acceptable pH (typically in the range of 4.0 to 10.5). Kit for use in the Production of the Radiotracer Compound

The present invention provides a kit for use in the production of a radiotracer compound of the first aspect, wherein the kit comprises two or more of the following components:

(i) a unit containing a non-radioactive precursor capable of reacting with

[ ¹⁸ F]-fluoride to produce the radiotracer compound described herein;

(ii) a unit containing a fluoride-extracting solid material for extracting fluoride from a solution;

(iii) a unit containing one of (a) a fluorosulfate-extracting solid material for

extracting fluorosulfate from a solution, (b) a hexafluorophosphate-extracting solid material for extracting hexafluorophosphate from a solution, or (c) a difluoromonophosphate-extracting solid material for extracting difluoromonophosphate from a solution; and

(iv) instructions for use of the kit in the production of a radiotracer compound of the first aspect.

In some embodiments, the kit comprises at least component (i). In some embodiments, the kit comprises at least three of components (i) to (iv). In one embodiment, the kit comprises component (i), component (ii), component (iii) and optionally component (iv). In a further embodiment, the kit comprises component (i), component (ii), component (iii) and component (iv).

In some embodiments, the kit further comprises one or more of the following components (units or vessels):

a unit containing a fluoride-retaining solid material for retaining [ ¹⁸ F]-fluoride before reaction of the [ ¹⁸ F]-fluoride with the non-radioactive precursor capable of reacting with [ ¹⁸ F]-fluoride;

a unit containing a fluoride-retaining solid material pre-conditioning solution for pre-conditioning the fluoride-retaining solid material;

a unit containing an fluoride-retaining solid material eluent for eluting the fluoride- retaining solid material;

a reaction vessel for reacting [ ¹⁸ F]-fluoride and the non-radioactive precursor capable of reacting with [ ¹⁸ F]-fluoride;

- a unit containing a fluoride-extracting solid material pre-conditioning solution for pre-conditioning the fluoride-extracting solid material;

a unit containing one of: (a) a fluorosulfate anion-exchanger solid material preconditioning solution for pre-conditioning the fluorosulfate-extracting solid material, (b) a hexafluorophosphate anion-exchanger solid material pre- conditioning solution for pre-conditioning the hexafluorophosphate-extracting solid material, or (c) a difluoromonophosphate anion-exchanger solid material pre-conditioning solution for pre-conditioning the difluoromonophosphate- extracting solid material;

a unit containing one of: (a) a fluorosulfate-extracting solid material eluent for eluting the fluorosulfate-extracting solid material (b) a hexafluorophosphate- extracting solid material eluent for eluting the hexafluorophosphate-extracting solid material, or (c) a difluoromonophosphate-extracting solid material eluent for eluting the difluoromonophosphate-extracting solid material; one or more units containing a solvent or a solvent mixture;

a unit containing one or more pharmaceutically acceptable carriers, excipients and/or biocompatible carriers; and

a collection vessel for receiving the radiotracer compound of the first aspect.

The kit may contain one or more reagent-containing units (such as units containing one or more reaction reagents, units containing a pre-conditioning solution and units containing an eluent solution), may contain one or more separation units (such as chromatography units for retaining or extracting species from a solution), and/or may contain instructions for use of the kit in the production of the radiotracer compound.

In one particular embodiment, the kit comprises the following components:

a unit containing a non-radioactive precursor capable of reacting with

[ ¹⁸ F]-fluoride to produce the radiotracer compound described herein; - a unit containing a fluoride-extracting solid material for extracting fluoride from a solution;

a unit containing a fluorosulfate-extracting solid material for extracting fluorosulfate from a solution;

a unit containing a fluoride-retaining solid material for retaining [ ¹⁸ F]-fluoride before reaction of the [ ¹⁸ F]-fluoride with the non-radioactive precursor capable of reacting with [ ¹⁸ F]-fluoride;

a unit containing a fluoride-retaining solid material pre-conditioning solution for pre-conditioning the fluoride-retaining solid material;

a unit containing an fluoride-retaining solid material eluent for eluting the fluoride-retaining solid material;

a reaction vessel for reacting [ ¹⁸ F]-fluoride and the non-radioactive precursor capable of reacting with [ ¹⁸ F]-fluoride; a unit containing a fluoride-extracting solid material pre-conditioning solution for pre-conditioning the fluoride-extracting solid material;

a unit containing a fluorosulfate anion-exchanger solid material pre-conditioning solution for pre-conditioning the fluorosulfate-extracting solid material; - a unit containing a fluorosulfate-extracting solid material eluent for eluting the fluorosulfate-extracting solid material; and

a collection vessel for receiving the radiotracer compound of the first aspect, and optionally the kit includes an automated synthesis cassette housing some or all of the components.

In another particular embodiment, the kit comprises the following components:

a unit containing a non-radioactive precursor capable of reacting with [ ¹⁸ F]-fluoride to produce the radiotracer compound described herein; a unit containing a fluoride-extracting solid material for extracting fluoride from a solution;

a unit containing a hexafluorophosphate-extracting solid material for extracting hexafluorophosphate from a solution;

a unit containing a fluoride-retaining material for retaining [ ¹⁸ F]-fluoride before reaction of the [ ¹⁸ F]-fluoride with the non-radioactive precursor capable of reacting with [ ¹⁸ F]-fluoride;

a unit containing a fluoride-retaining material pre-conditioning solution for pre-conditioning the fluoride-retaining material;

a unit containing an fluoride-retaining material eluent for eluting the

fluoride-retaining material;

- a reaction vessel for reacting [ ¹⁸ F]-fluoride and the non-radioactive precursor capable of reacting with [ ¹⁸ F]-fluoride;

a unit containing a fluoride-extracting solid material pre-conditioning solution for pre-conditioning the fluoride-extracting solid material; a unit containing a hexafluorophosphate anion-exchanger solid material preconditioning solution for pre-conditioning the hexafluorophosphate-extracting solid material;

a unit containing a hexafluorophosphate-extracting solid material eluent for eluting the hexafluorophosphate-extracting solid material; and

a collection vessel for receiving the radiotracer compound of the first aspect, and optionally the kit includes an automated synthesis cassette housing some or all of the components. In a further particular embodiment, the kit comprises the following components:

a unit containing a non-radioactive precursor capable of reacting with [ ¹⁸ F]-fluoride to produce the radiotracer compound described herein; a unit containing a fluoride-extracting solid material for extracting fluoride from a solution;

- a unit containing a difluoromonophosphate-extracting solid material for extracting difluoromonophosphate from a solution;

a unit containing a fluoride-retaining material for retaining [ ¹⁸ F]-fluoride before reaction of the [ ¹⁸ F]-fluoride with the non-radioactive precursor capable of reacting with [ ¹⁸ F]-fluoride;

- a unit containing a fluoride-retaining material pre-conditioning solution for

pre-conditioning the fluoride-retaining material;

a unit containing an fluoride-retaining material eluent for eluting the

fluoride-retaining material;

a reaction vessel for reacting [ ¹⁸ F]-fluoride and the non-radioactive precursor capable of reacting with [ ¹⁸ F]-fluoride;

a unit containing a fluoride-extracting solid material pre-conditioning solution for pre-conditioning the fluoride-extracting solid material; a unit containing a difluoromonophosphate anion-exchanger solid material preconditioning solution for pre-conditioning the difluoromonophosphate-extracting solid material;

a unit containing a difluoromonophosphate-extracting solid material eluent for eluting the difluoromonophosphate-extracting solid material; and

a collection vessel for receiving the radiotracer compound of the first aspect, and optionally the kit includes an automated synthesis cassette housing some or all of the components. In some embodiments, a unit or vessel listed above may act as more than one component in the kit. For example, the reaction vessel may be preloaded with the non-radioactive precursor capable of reacting with [ ¹⁸ F]-fluoride. In other words, that the reaction vessel and the unit containing the non-radioactive precursor capable of reacting with [ ¹⁸ F]-fluoride are the same component. Alternatively, the unit containing the fluoride-retaining solid material may also form the reaction vessel. Additionally or alternatively, the unit containing the fluoride-retaining solid material may also form the unit containing the fluoride-extracting solid material.

Automated Synthesis Cassette

In some embodiments, the unit(s) and/or vessel(s) of the kit are housed in a cassette adapted to be used with an automated radiotracer synthesizer apparatus. The cassette may be adapted to fit interchangeably onto an automated synthesizer apparatus. Automated synthesizer apparatuses are known and commercially available from a number of suppliers such as GE Healthcare, CTI Inc, Ion Beam Applications S.A. (Belgium), Raytest (Germany) and Bioscan (USA).

Automated synthesis cassettes are known per se. The cassette typically includes a slot or position within the cassette to accommodate each unit or vessel required for the automated synthesis. The cassette may include an array of valves with each valve linked to a port where units and/or vessels of the kit and/or additional reagents or vials may be attached. The cassette may have any number of valves. The cassette may have 15 to 40 valves, 20 to 30 or around 25 valves. 3-way valves may be used. The cassette may further include one or more conduits (such as tubes) in fluid connection between one or more units, valves, ports and/or vessels of the cassette for transfer of fluid to or from a unit or vessel of the cassette. Furthermore, the cassette may be adapted such that mechanical movement of moving parts of the synthesizer controls the operation of the cassette from outside of the cassette.

In some embodiments, the cassette includes a reaction vessel, a plurality of ports to connect to one or more reagent-containing units and/or one or more separation units of the kit, a port to connect to a collection vessel, a solvent inlet port for receiving a solvent or solvent mixture, and a waste outlet port for disposing of waste from the cassette. In some embodiments, the cassette includes all of the reagent-containing units and/or one or more separation units of the kit required to produce the radiotracer compound described herein with the exception of the radioactive [ ¹⁸ F] precursor. In such embodiments, the radioactive [ ¹⁸ F] precursor and any solvents may typically be introduced into the cassette from an external source through one or more inlet ports.

Method of Using Automatic Synthesis Cassette

When the kit includes a cassette, the kit can be used in a method with an automatic radiotracer synthesizer. A method of using the cassette with an automatic radiotracer synthesizer will now be described.

The cassette may be fitted onto the automatic synthesizer, including coupling all required fittings between the synthesizer and the cassette. A user may initiate an automated synthesis protocol on the automatic synthesizer. The protocol may start with a fluoride-retaining solid material pre-conditioning step. A fluoride-retaining solid material pre-conditioning solution may be transferred from a unit containing a fluoride-retaining solid material pre-conditioning solution through a tube to an inlet of a unit containing fluoride-retaining solid material. The fluoride-retaining solid material pre-conditioning solution may pass over the fluoride-retaining solid material before leaving the unit containing a fluoride-retaining solid material though an outlet. The used

fluoride-retaining solid material pre-conditioning solution may be disposed of into a waste unit. A solvent, such as water, may then be transferred from a unit containing the solvent through a tube into the unit containing the fluoride-retaining solid material. The solvent may pass over the fluoride-retaining solid material before leaving the unit containing a

fluoride-retaining solid material though an outlet. The used solvent may be disposed of into a waste unit. The automatic protocol may include a [ ¹⁸ F]-fluoride-retaining and eluting step. This step may follow the fluoride-retaining solid material pre-conditioning step. A solution of [ ¹⁸ F]-fluoride may be introduced into the inlet of the unit containing fluoride-retaining solid material. The solution of [ ¹⁸ F]-fluoride may pass over the fluoride-retaining solid material so that some [ ¹⁸ F]-fluoride is retained by the fluoride-retaining solid material. The used [ ¹⁸ F]-fluoride solution may be disposed of into a radioactive waste unit. A fluoride-retaining solid material eluent may then be transferred from a unit containing the fluoride-retaining solid material eluent through a tube into the unit containing the fluoride-retaining solid material. The fluoride-retaining solid material eluent may pass over the fluoride-retaining solid material to release [ ¹⁸ F]-fluoride into the eluent, before leaving the unit containing a fluoride-retaining solid material though an outlet. The eluted eluent containing [ ¹⁸ F]-fluoride may be transferred to a reaction vessel. The automatic protocol may include a reaction step. In the reaction step, [ ¹⁸ F]-fluoride may be transferred to the reaction vessel. In addition, the non-radioactive precursor may be transferred from a unit containing the non-radioactive precursor to the reaction vessel. In alternative embodiments, the non-radioactive precursor may be pre-loaded in the reaction vessel (for example where the reaction vessel and the unit containing the non-radioactive precursor are the same component). In a further alternative embodiment, the unit containing the fluoride-retaining solid material may act as the reaction vessel if the [ ¹⁸ F]-fluoride is not eluted from the unit. In this embodiment non-radioactive precursor may be transferred from a unit containing the non-radioactive precursor to the unit containing the fluoride-retaining solid material (acting as the reaction vessel).

Additional reagents and/or solvent may also be transferred to the reaction vessel. The reaction vessel then allows the reaction to occur, for example, with stirring and/or heating of the reaction mixture. The reaction mixture may be quenched, for example, by the transfer of a solvent, such as water, into the reaction vessel.

The automatic protocol may include a purification step. The purification step may include passing the reacted reaction mixture over a fluoride-extracting solid material and/or one of:

(a) a fluorosulfate-extracting solid material, (b) a hexafluorophosphate-extracting material, or (c) a difluoromonophosphate-extracting material. In particular embodiments, the purification step includes passing the reacted reaction mixture over a fluoride-extracting solid material and then passing the eluted solution over one of (a) a fluorosulfate-extracting solid material,

(b) a hexafluorophosphate-extracting material, or (c) a difluoromonophosphate-extracting material. The choice of extracting material will depend on which of [ ¹⁸ F]S03F ^" , [ ¹⁸ F]PF6 ^" or [ ¹⁸ F]P02F ₂ ^" is being synthesised.

The fluoride-extracting solid material may be preconditioned by transferring a

fluoride-extracting solid material preconditioning solution from a unit containing a fluoride-extracting solid material preconditioning solution into an inlet of the unit containing the a fluoride-extracting solid material, passing the preconditioning solution over the solid material, before the used pre-conditioning solution leaves the unit through an outlet. The used pre-conditioning solution may be disposed of in a waste unit. A solvent, such as water, or a gas, such as air, may then be transferred from a unit containing the solvent or gas through a tube into the unit containing the fluoride-extracting solid material. The solvent or gas may pass over the fluoride-extracting solid material before leaving the unit containing a fluoride-extracting solid material though an outlet. The used solvent or gas may be disposed of into a waste unit.

The reacted reaction mixture may be transferred to the inlet of the unit containing the fluoride-extracting solid material and passed over the fluoride-extracting solid material to remove unreacted fluoride.

The eluted reaction mixture may then leave the unit through an outlet and be transferred to another purification unit (such as the unit containing one of the fluorosulfate-extracting solid material, hexafluorophosphate-extracting solid material or difluoromonophosphate-extracting solid material) or to a collection vessel. Purification of r ¹⁸ FlS03F-

The fluorosulfate-extracting solid material may be preconditioned by transferring a fluorosulfate-extracting solid material preconditioning solution from a unit containing a fluorosulfate-extracting solid material preconditioning solution into an inlet of the unit containing the a fluorosulfate-extracting solid material, passing the preconditioning solution over the solid material, before the used pre-conditioning solution leaves the unit through an outlet. The used pre-conditioning solution may be disposed of in a waste unit. A solvent, such as water, may then be transferred from a unit containing the solvent through a tube into the unit containing the fluorosulfate-extracting solid material. The solvent may pass over the fluorosulfate-extracting solid material before leaving the unit containing a fluorosulfate-extracting solid material though an outlet. The used solvent or gas may be disposed of into a waste unit. The reacted reaction mixture may be transferred to the inlet of the unit containing the fluorosulfate-extracting solid material and passed over the fluorosulfate-extracting solid material to extract the [ ¹⁸ F]-fluorosulfate. The eluted reaction mixture may then leave the unit through an outlet and be transferred to the same purification unit (for one or more passes over the fluorosulfate-extracting solid material to extract more of the

[ ¹⁸ F]-fluorosulfate), another purification unit (such as another unit containing

fluorosulfate-extracting solid material) or to a waste unit.

A fluorosulfate extracting solid material eluent may then be transferred to the inlet of the unit containing the fluorosulfate-extracting solid material and passed over the

fluorosulfate-extracting solid material to elute the [ ¹⁸ F]-fluorosulfate. The eluted fluorosulfate- extracting solid material eluent (containing [ ¹⁸ F]-fluorosulfate) may then leave the unit through an outlet and be transferred to the same purification unit (for one or more passes over the fluorosulfate-extracting solid material to elute more of the [ ¹⁸ F]-fluorosulfate), another purification unit (such as another unit containing fluoride-extracting solid material) or to a collection vessel.

Purification of r ¹⁸ FlPF^

The hexafluorophosphate-extracting solid material may be preconditioned by transferring a hexafluorophosphate-extracting solid material preconditioning solution from a unit containing a hexafluorophosphate-extracting solid material preconditioning solution into an inlet of the unit containing the a hexafluorophosphate-extracting solid material, passing the

preconditioning solution over the solid material, before the used pre-conditioning solution leaves the unit through an outlet. The used pre-conditioning solution may be disposed of in a waste unit. A solvent, such as water, may then be transferred from a unit containing the solvent through a tube into the unit containing the hexafluorophosphate-extracting solid material. The solvent may pass over the hexafluorophosphate-extracting solid material before leaving the unit containing a hexafluorophosphate-extracting solid material though an outlet. The used solvent or gas may be disposed of into a waste unit.

The reacted reaction mixture may be transferred to the inlet of the unit containing the hexafluorophosphate-extracting solid material and passed over the

hexafluorophosphate-extracting solid material to extract the [ ¹⁸ F]-hexafluorophosphate. The eluted reaction mixture may then leave the unit through an outlet and be transferred to the same purification unit (for one or more passes over the hexafluorophosphate-extracting solid material to extract more of the [ ¹⁸ F]-hexafluorophosphate), another purification unit (such as another unit containing hexafluorophosphate-extracting solid material) or to a waste unit. A hexafluorophosphate extracting material eluent may then be transferred to the inlet of the unit containing the hexafluorophosphate-extracting solid material and passed over the hexafluorophosphate-extracting solid material to elute the [ ¹⁸ F]-hexafluorophosphate. The eluted hexafluorophosphate-extracting material eluent (containing [ ¹⁸ F]- hexafluorophosphate) may then leave the unit through an outlet and be transferred to the same purification unit (for one or more passes over the hexafluorophosphate-extracting solid material to elute more of the [ ¹⁸ F]-hexafluorophosphate), another purification unit (such as another unit containing fluoride-extracting solid material) or to a collection vessel.

Purification of r ¹⁸ FlPO?F

The difluoromonophosphate-extracting solid material may be preconditioned by transferring a difluoromonophosphate-extracting solid material preconditioning solution from a unit containing a difluoromonophosphate-extracting solid material preconditioning solution into an inlet of the unit containing the a difluoromonophosphate-extracting solid material, passing the preconditioning solution over the solid material, before the used pre-conditioning solution leaves the unit through an outlet. The used pre-conditioning solution may be disposed of in a waste unit. A solvent, such as water, may then be transferred from a unit containing the solvent through a tube into the unit containing the difluoromonophosphate-extracting solid material. The solvent may pass over the difluoromonophosphate-extracting solid material before leaving the unit containing a difluoromonophosphate-extracting solid material though an outlet. The used solvent or gas may be disposed of into a waste unit.

The reacted reaction mixture may be transferred to the inlet of the unit containing the difluoromonophosphate-extracting solid material and passed over the

difluoromonophosphate-extracting solid material to extract the [ ¹⁸ F]-difluoromonophosphate.

The eluted reaction mixture may then leave the unit through an outlet and be transferred to the same purification unit (for one or more passes over the

difluoromonophosphate-extracting solid material to extract more of the

[ ¹⁸ F]-difluoromonophosphate), another purification unit (such as another unit containing difluoromonophosphate-extracting solid material) or to a waste unit.

A difluoromonophosphate extracting material eluent may then be transferred to the inlet of the unit containing the difluoromonophosphate-extracting solid material and passed over the difluoromonophosphate-extracting solid material to elute the [ ¹⁸ F]-difluoromonophosphate. The eluted difluoromonophosphate-extracting material eluent (containing

[ ¹⁸ F]-difluoromonophosphate) may then leave the unit through an outlet and be transferred to the same purification unit (for one or more passes over the

difluoromonophosphate-extracting solid material to elute more of the

[ ¹⁸ F]-difluoromonophosphate), another purification unit (such as another unit containing fluoride-extracting solid material) or to a collection vessel. One or more solvents and/or one or more pharmaceutically acceptable carriers, excipients and/or biocompatible carriers may be transferred before or after the radiotracer compound as described herein is transferred to the collection vessel. The collection vessel may contain the radiotracer compound in a composition suitable for administration to a subject. In such embodiments, the collection vessel may be a syringe for administering the radiotracer composition to a subject.

Reagent-Containing Unit(s)

The kit may include one or more reagent containing units selected from the group consisting of: a unit containing a non-radioactive precursor capable of reacting with [ ¹⁸ F]-fluoride to produce the radiotracer compound described herein; a unit containing a fluoride-retaining solid material pre-conditioning solution for pre conditioning the fluoride-retaining solid material; a unit containing an fluoride-retaining solid material eluent for eluting the fluoride- retaining solid material; a unit containing a fluoride-extracting solid material pre- conditioning solution for pre-conditioning the fluoride-extracting solid material; a unit containing a fluorosulfate-extracting solid material pre-conditioning solution for preconditioning the fluorosulfate-extracting solid material; a unit containing a fluorosulfate- extracting solid material eluent for eluting the fluorosulfate-extracting solid material; and a unit containing one or more pharmaceutically acceptable carriers, excipients and/or biocompatible carriers.

One or more of the reagent-containing units may include a sealed vial or syringe containing the reagent. The seal may be, for example, a septum seal or one half of a marrying joint. The other half of the marrying joint may be formed by a port in an automated synthesis cassette. [ ¹⁸ F1SQ3F ^' reagent-containing units

The unit containing a non-radioactive precursor capable of reacting with [ ¹⁸ F]-fluoride to produce the radiotracer compound described herein may contain SO3 gas or a solution of an adduct of SO3. The adduct of SO3 may be a Lewis adduct of a Lewis base and SO3 as described herein. The kit may further include a conduit (such as a plastic tube) connecting this unit to the reaction vessel and/or one or more units containing a solvent or a solvent mixture, when present.

In some embodiments, the unit containing the non-radioactive precursor capable of reacting with [ ¹⁸ F]-fluoride may include the Lewis adduct covalently bonded to a solid material. Such solid material may be, for example, a polymer or silica. The Lewis adduct may be covalently bonded to the solid material through the Lewis base. In other words, the unit containing the non-radioactive precursor capable of reacting may be a column or cartridge including a Lewis adduct of a Lewis base and SO3 covalently bonded to solid material through the Lewis base. Such a unit may be used as the reaction vessel in the kit by passing [ ¹⁸ F]-fluoride solution over the solid phase material. As a result, the radiotracer compound (including [ ¹⁸ F]-fluorosulfate) may be synthesised in the unit and elute from the unit. Unreacted Lewis adduct may be left behind in the unit. [ ¹⁸ F1PF6 ^" reagent-containing units

The unit containing a non-radioactive precursor capable of reacting with [ ¹⁸ F]-fluoride to produce the radiotracer compound described herein may contain PF ₅ gas, PCI5 or a solution of an adduct of PF ₅ . The adduct of PF ₅ may be a Lewis adduct of a Lewis base and PF ₅ as described herein. The kit may further include a conduit (such as a plastic tube) connecting this unit to the reaction vessel and/or one or more units containing a solvent or a solvent mixture, when present. In some embodiments, the unit containing the non-radioactive precursor capable of reacting with [ ¹⁸ F]-fluoride may include the Lewis adduct covalently bonded to a solid material. Such solid material may be, for example, a polymer or silica. The Lewis adduct may be covalently bonded to the solid material through the Lewis base. In other words, the unit containing the non-radioactive precursor capable of reacting may be a column or cartridge including a

Lewis adduct of a Lewis base and PF ₅ covalently bonded to solid material through the Lewis base. Such a unit may be used as the reaction vessel in the kit by passing [ ¹⁸ F]-fluoride solution over the solid phase material. As a result, the radiotracer compound (including [ ¹⁸ F]-hexafluorophosphate) may be synthesised in the unit and elute from the unit.

Unreacted Lewis adduct may be left behind in the unit.

[ ¹⁸ F1PQ2F2 ^" reagent-containing units

The unit containing a non-radioactive precursor capable of reacting with [ ¹⁸ F]-fluoride to produce the radiotracer compound described herein may contain PF ₅ gas, PCIs or a solution of an adduct of PO2F. The adduct of PO2F may be a Lewis adduct of a Lewis base and PO2F as described herein. The kit may further include a conduit (such as a plastic tube) connecting this unit to the reaction vessel and/or one or more units containing a solvent or a solvent mixture, when present. In some embodiments, the unit containing the non-radioactive precursor capable of reacting with [ ¹⁸ F]-fluoride may include the Lewis adduct covalently bonded to a solid material. Such solid material may be, for example, a polymer or silica. The Lewis adduct may be covalently bonded to the solid material through the Lewis base. In other words, the unit containing the non-radioactive precursor capable of reacting may be a column or cartridge including a Lewis adduct of a Lewis base and PO2F covalently bonded to solid material through the Lewis base. Such a unit may be used as the reaction vessel in the kit by passing [ ¹⁸ F]- fluoride solution over the solid phase material. As a result, the radiotracer compound (including [ ¹⁸ F]-difluoromophosphate) may be synthesised in the unit and elute from the unit. Unreacted Lewis adduct may be left behind in the unit.

The unit containing a fluoride-retaining solid material pre-conditioning solution may be used for pre-conditioning the fluoride-retaining solid material. The fluoride-retaining solid material pre-conditioning solution may be a solution of a Bransted base, such as KHCO3, K2CO3, NaHCC>3, Na2CC>3, NH4HCO3, and (NH ₄ )2CC>3. In some embodiments, the solution is an aqueous solution. The kit may further include one or more conduits (such as a plastic tube) connecting this unit to other vessels and/or units of the kit, such as the unit a fluoride- retaining solid material and/or one or more units containing a solvent or a solvent mixture, when present.

The unit containing a fluoride-retaining solid material eluent may be used for eluting the fluoride-retaining solid material. The eluent may be a solution containing a Bransted base, such as KHCO3, K2CO3, NaHCOs, Na ₂ C0 ₃ , NH4HCO3, and (NH ₄ ) ₂ C0 ₃ and/or a cryptand, such as Kryptofix 2.2.2 (K222). The kit may further include one or more conduits (such as a plastic tube) connecting this unit to other vessels and/or units of the kit, such as the unit containing a fluoride-retaining solid material, and/or one or more units containing a solvent or a solvent mixture, when present.

The unit containing a fluoride-extracting solid material pre-conditioning solution may be used for pre-conditioning the fluoride-extracting solid material. In some embodiments, the fluoride-extracting solid material pre-conditioning solution is a solvent, such as water.

Accordingly, the unit containing a fluoride-extracting solid material pre-conditioning solution may form a unit containing a solvent or a solvent mixture. The kit may further include one or more conduits (such as a plastic tube) connecting this unit to other vessels and/or units of the kit, such as the present unit containing a fluoride-extracting solid material, when present. The unit containing a fluorosulfate anion-exchanger solid material pre-conditioning solution may be used for pre-conditioning the fluorosulfate anion-exchanger solid material. The fluorosulfate anion-exchanger solid material pre-conditioning solution may contain one or more salts such as NaCI or KCI. The concentration of the salt in the fluorosulfate

anion-exchanger solid material pre-conditioning solution is not particularly limited, but may be in the range of 0.2 to 3 M, 0.5 to 2 M or 0.8 to 1 .2 M. The fluorosulfate anion-exchanger solid material pre-conditioning solution may be an aqueous solution. In one embodiment, the fluorosulfate anion-exchanger solid material pre-conditioning solution is an aqueous NaCI or KCI solution with a salt concentration in the range of 0.8 to 1 .2 M. The kit may further include one or more conduits (such as a plastic tube) connecting this unit to other vessels and/or units of the kit, such as the reaction vessel, the unit containing a fluoride- extracting solid material, one or more units containing a solvent or a solvent mixture and/or the collecting vessel, when present. The unit containing a fluorosulfate anion-exchanger solid material eluent may be used for eluting the fluorosulfate anion-exchanger solid material. The fluorosulfate anion-exchanger solid material eluent may be a NaCI solution or a buffer solution. The buffer solution may be any solution with a sufficient ionic content to elute the radiotracer compound. Alternatively, if a weak anion exchanger (WAX or Nh ) is used as the fluorosulfate anion-exchanger solid material, the fluorosulfate anion-exchanger solid material eluent may be a basic solution. Such a basic solution increases the pH to cancel the anion binding properties of the fluorosulfate anion-exchanger solid material and elute the product. The kit may further include one or more conduits (such as a plastic tube) connecting this unit to other vessels and/or units of the kit, such as the unit containing a fluorosulfate anion exchange solid material, and/or one or more units containing a solvent or a solvent mixture, when present.

The unit containing a fluoride-extracting solid material pre-conditioning solution may be used for pre-conditioning the fluoride-extracting solid material. In some embodiments, the fluoride-extracting solid material pre-conditioning solution is a solvent, such as water.

Accordingly, the unit containing a fluoride-extracting solid material pre-conditioning solution may form a unit containing a solvent or a solvent mixture. The kit may further include one or more conduits (such as a plastic tube) connecting this unit to other vessels and/or units of the kit, such as the unit containing a fluoride-extracting solid material, when present.

The unit containing a hexafluorophosphate anion-exchanger solid material pre-conditioning solution may be used for pre-conditioning the hexafluorophosphate anion-exchanger solid material. The hexafluorophosphate anion-exchanger solid material pre-conditioning solution may contain one or more salts such as NaCI or KCI. The concentration of the salt in the hexafluorophosphate anion-exchanger solid material pre-conditioning solution is not particularly limited, but may be in the range of 0.2 to 3 M, 0.5 to 2 M or 0.8 to 1 .2 M. The hexafluorophosphate anion-exchanger solid material pre-conditioning solution may be an aqueous solution. In one embodiment, the hexafluorophosphate anion-exchanger solid material pre-conditioning solution is an aqueous NaCI or KCI solution with a salt

concentration in the range of 0.8 to 1 .2 M. The kit may further include one or more conduits (such as a plastic tube) connecting this unit to other vessels and/or units of the kit, such as the reaction vessel, the unit containing a fluoride-extracting solid material, one or more units containing a solvent or a solvent mixture and/or the collecting vessel, when present.

The unit containing a hexafluorophosphate anion-exchanger solid material eluent may be used for eluting the hexafluorophosphate anion-exchanger solid material. The

hexafluorophosphate anion-exchanger solid material eluent may be a NaCI solution or a buffer solution. The buffer solution may be any solution with a sufficient ionic content to elute the radiotracer compound. Alternatively, if a weak anion exchanger (WAX or Nh ) is used as the hexafluorophosphate anion-exchanger solid material, the hexafluorophosphate anion-exchanger solid material eluent may be a basic solution. Such a basic solution increases the pH to cancel the anion binding properties of the hexafluorophosphate anion-exchanger solid material and elute the product. The kit may further include one or more conduits (such as a plastic tube) connecting this unit to other vessels and/or units of the kit, such as the unit containing a hexafluorophosphate anion exchange solid material, and/or one or more units containing a solvent or a solvent mixture, when present.

The unit containing a difluoromonophosphate anion-exchanger solid material preconditioning solution may be used for pre-conditioning the difluoromonophosphate anion-exchanger solid material. The difluoromonophosphate anion-exchanger solid material pre-conditioning solution may contain one or more salts such as NaCI or KCI. The concentration of the salt in the difluoromonophosphate anion-exchanger solid material preconditioning solution is not particularly limited, but may be in the range of 0.2 to 3 M, 0.5 to 2 M or 0.8 to 1.2 M. The difluoromonophosphate anion-exchanger solid material preconditioning solution may be an aqueous solution. In one embodiment, the

difluoromonophosphate anion-exchanger solid material pre-conditioning solution is an aqueous NaCI or KCI solution with a salt concentration in the range of 0.8 to 1 .2 M. The kit may further include one or more conduits (such as a plastic tube) connecting this unit to other vessels and/or units of the kit, such as the reaction vessel, the unit containing a fluoride-extracting solid material, one or more units containing a solvent or a solvent mixture and/or the collecting vessel, when present.

The unit containing a difluoromonophosphate anion-exchanger solid material eluent may be used for eluting the difluoromonophosphate anion-exchanger solid material. The

difluoromonophosphate anion-exchanger solid material eluent may be a NaCI solution or a buffer solution. The buffer solution may be any solution with a sufficient ionic content to elute the radiotracer compound. Alternatively, if a weak anion exchanger (WAX or Nh ) is used as the difluoromonophosphate anion-exchanger solid material, the

difluoromonophosphate anion-exchanger solid material eluent may be a basic solution. Such a basic solution increases the pH to cancel the anion binding properties of the difluoromonophosphate anion-exchanger solid material and elute the product. The kit may further include one or more conduits (such as a plastic tube) connecting this unit to other vessels and/or units of the kit, such as the unit containing a difluoromonophosphate anion exchange solid material, and/or one or more units containing a solvent or a solvent mixture, when present.

The kit may include a unit containing one or more pharmaceutically acceptable carriers, excipients and/or biocompatible carriers for adding to the radiotracer compound to form an a radiotracer composition in a form suitable for administration to a subject. The kit may further include one or more conduits (such as a plastic tube) connecting this unit to other vessels and/or units of the kit, such as the collection vessel.

Separation Unit(s)

The kit may comprise one or more separation units selected from the group consisting of: a unit containing a fluoride-retaining solid material; a unit containing a fluoride-extracting solid material; and unit containing a fluorosulfate-extracting solid material.

One or more of the separation units may include a cartridge housing containing a separating solid material (such as a fluoride-retaining solid material, a fluoride-extracting solid material or a fluorosulfate-extracting solid material). Such a cartridge is typically adapted to be used with an automated synthesis cassette.

The kit may comprise one or more separation units selected from the group consisting of: a unit containing a fluoride-retaining solid material; a unit containing a fluoride-extracting solid material; and unit containing a hexafluorophosphate-extracting solid material.

One or more of the separation units may include a cartridge housing containing a separating solid material (such as a fluoride-retaining solid material, a fluoride-extracting solid material or a hexafluorophosphate-extracting solid material). Such a cartridge is typically adapted to be used with an automated synthesis cassette.

The kit may comprise one or more separation units selected from the group consisting of: a unit containing a fluoride-retaining solid material; a unit containing a fluoride-extracting solid material; and unit containing a difluoromonophosphate-extracting solid material.

One or more of the separation units may include a cartridge housing containing a separating solid material (such as a fluoride-retaining solid material, a fluoride-extracting solid material or a difluoromonophosphate-extracting solid material). Such a cartridge is typically adapted to be used with an automated synthesis cassette.

The unit containing a fluoride-retaining solid material may be used for retaining [ ¹⁸ F]-fluoride before reaction with the non-radioactive precursor capable of reacting with [ ¹⁸ F]-fluoride. The fluoride-retaining solid material may an anion-exchange solid material. For example, the fluoride-retaining solid material may be an aluminium-containing mineral or a

calcium-containing mineral. In some embodiments, the unit containing the

fluoride-extracting solid material is selected from the group consisting of an alumina column, an aluminium hydroxide column and a hydroxyapatite column. The kit may further include one or more conduits (such as a plastic tube) connecting this unit to other vessels and/or units of the kit, such as the unit containing a fluoride-retaining solid material pre-conditioning solution, the unit containing a fluoride-retaining solid material eluent, one or more units containing a solvent or a solvent mixture and/or the reaction vessel, when present. In some embodiments, the unit containing a fluoride-retaining solid material may be used as the reaction vessel of the kit when [ ¹⁸ F]-fluoride is retained on the fluoride-retaining solid material. A solution of the non-radioactive precursor capable of reacting with [ ¹⁸ F]-fluoride may be passed over the fluoride-retaining solid material with retained [ ¹⁸ F]-fluoride to produce the radiotracer compound described herein, for example, in the unit or in the solution passing through the unit.

The unit containing a fluoride-extracting solid material for extracting fluoride from a solution can be used as a purification unit for removing unreacted fluoride from a reaction mixture. As described above, the fluoride-extracting solid material is any material capable of extracting fluoride (such as unreacted [ ¹⁸ F]F ^" ) from a solution. The fluoride-extracting solid material may be an aluminium-containing mineral or a calcium-containing mineral. In some embodiments, the fluoride-extracting solid material is selected from the group consisting of an alumina column, an aluminium hydroxide column and a hydroxyapatite column. The kit may further include one or more conduits (such as a plastic tube) connecting this unit to other vessels and/or units of the kit, such as the reaction vessel, the unit containing fluorosulfate anion-exchange solid material, one or more units containing a solvent or a solvent mixture and/or the collecting vessel, when present.

In some embodiments, the unit containing the fluoride-retaining solid material may also act as the unit containing the fluoride-extracting solid material. The unit may be used to retain fluoride, and then used to extract fluoride with optional washes with solvent, eluting solutions and/or preconditioning solutions between uses.

The unit containing a fluorosulfate-extracting solid material for extracting fluorosulfate from a solution is a purification unit for retaining the radiotracer compound. As described above, the fluorosulfate-extracting solid material may be any material capable of extracting fluorosulfate from a solution, such as an anion-exchanger solid material. The

fluorosulfate-extracting solid material may be a silica-based, hydrophilic, strong

anion-exchanger (such as a SAX, strong anion exchanger with quaternary ammonium functional group, e.g. tetraalkylammonium-derivatised silica;) or a weak anion exchanger (such as a WAX, a weak anion exchanger with a tertiary amine functional group, or a primary amine derivatised silica). Such anion exchangers are known and widely available. The pore size of the anion exchanger is not limited. In some embodiments, the pore size of the anion exchanger is in the range of 200 to 400 A. In some embodiments, the unit containing the fluorosulfate anion exchanger solid material is a QMA cartridge or a SAX cartridge (strong anion exchanger with quaternary ammonium functional group). The kit may further include one or more conduits (such as a plastic tube) connecting this unit to other vessels and/or units of the kit, such as the reaction vessel, the unit containing a fluoride- extracting solid material, one or more units containing a solvent or a solvent mixture and/or the collecting vessel, when present.

The unit containing a hexafluorophosphate-extracting solid material for extracting hexafluorophosphate from a solution is a purification unit for retaining the radiotracer compound. As described above, the hexafluorophosphate-extracting solid material may be any material capable of extracting hexafluorophosphate from a solution, such as an anion-exchanger solid material. The hexafluorophosphate-extracting solid material may be a silica-based, hydrophilic, strong anion-exchanger (such as a SAX, strong anion exchanger with quaternary ammonium functional group, e.g. tetraalkylammonium-derivatised silica;) or a weak anion exchanger (such as a WAX, a weak anion exchanger with a tertiary amine functional group, or a primary amine derivatised silica). Such anion exchangers are known and widely available. The pore size of the anion exchanger is not limited. In some embodiments, the pore size of the anion exchanger is in the range of 200 to 400 A. In some embodiments, the unit containing the hexafluorophosphate-extracting solid material is a QMA cartridge or a SAX cartridge (strong anion exchanger with quaternary ammonium functional group). The kit may further include one or more conduits (such as a plastic tube) connecting this unit to other vessels and/or units of the kit, such as the reaction vessel, the unit containing a fluoride-extracting solid material, one or more units containing a solvent or a solvent mixture and/or the collecting vessel, when present. The unit containing a difluoromonophosphate-extracting solid material for extracting difluoromonophosphate from a solution is a purification unit for retaining the radiotracer compound. As described above, the difluoromonophosphate-extracting solid material may be any material capable of extracting difluoromonophosphate from a solution, such as an anion-exchanger solid material. The difluoromonophosphate-extracting solid material may be a silica-based, hydrophilic, strong anion-exchanger (such as a SAX, strong anion exchanger with quaternary ammonium functional group, e.g. tetraalkylammonium-derivatised silica;) or a weak anion exchanger (such as a WAX, a weak anion exchanger with a tertiary amine functional group, or a primary amine derivatised silica). Such anion exchangers are known and widely available. The pore size of the anion exchanger is not limited. In some embodiments, the pore size of the anion exchanger is in the range of 200 to 400 A. In some embodiments, the unit containing the difluoromonophosphate-extracting solid material is a QMA cartridge or a SAX cartridge (strong anion exchanger with quaternary ammonium functional group). The kit may further include one or more conduits (such as a plastic tube) connecting this unit to other vessels and/or units of the kit, such as the reaction vessel, the unit containing a fluoride-extracting solid material, one or more units containing a solvent or a solvent mixture and/or the collecting vessel, when present.

Each of the separation unit may be used once to provide a single use unit. Alternatively, one or more of the separation units may be use more than once to synthesise the radiotracer compound described herein, with optional washes with solvent, eluting solutions and/or pre-conditioning solutions, as appropriate.

Vessel(s)

The kit may comprise one or more vessels selected from the group consisting of: a reaction vessel, and a collection vessel. The reaction vessel may be used for reacting [ ¹⁸ F]-fluoride and the non-radioactive precursor capable of reacting with [ ¹⁸ F]-fluoride. The reaction vessel may include a stirring mechanism for stirring the reaction mixture. The reaction vessel may include a heating element to heat the reaction mixture. The size of the reaction vessel is not particularly limited. The reaction vessel may have a volume of around 1 to 10 cm ³ . The kit may further include one or more conduits (such as a plastic tube) connecting the reaction vessel to other vessels and/or units of the kit, such as the unit containing a fluoride-retaining solid material, the unit containing a non-radioactive precursor capable of reacting with [ ¹⁸ F]-fluoride to produce the radiotracer compound described herein, the unit containing a fluoride-extracting solid material, the unit containing one of (a) a fluorosulfate anion-exchanger solid material, (b) hexafluorophosphate anion-exchanger solid material or (c) difluoromonophosphate anion-exchanger solid material, and/or one or more units containing a solvent or a solvent mixture, when present.

The collection vessel may be used for receiving the radiotracer compound of the first aspect. The collection vessel may be a vial or a syringe.

Solvent-Containing Unit(s)

The kit may include one or more units containing a solvent or a solvent mixture. The type of solvent is not limited and the kit can include any number of units containing a solvent or mixture of solvents. The solvent may be used for any purpose in the kit, such as

preconditioning units before use, adding solvent to the reaction vessel, washing or flushing conduits (e.g. tubes) connecting units and/or vessels of the kit before and/or after transfer of fluid through the conduit, and washing units after use. Typical solvents include water, acetonitrile and mixtures thereof. The kit may further include one or more conduits (such as a plastic tube) connecting this unit to other vessels and/or units of the kit.

Each of the reagent-containing unit of the kit may contain enough reagent for one synthesis or multiple syntheses. Accordingly, the kit may be a single use kit or a multiple use kit. Each of the separation units may be adapted for single use or multiple uses. In some embodiments, the kit is a single-use disposable cassette including one or more

reagent-containing units and one or more separation units adapted for single use. Use of the Radiotracer Compound and Radiotracer Compound Composition

The radiotracer compounds and radiotracer compositions described herein have particular use in diagnostic medical imaging methods, in particular PET imaging. The present invention provides a radiotracer compound or radiotracer composition as described herein for use in a diagnostic method of medical imaging. In some embodiments, the diagnostic method of medical imaging is PET imaging, optionally in combination with computed tomography (CT) or magnetic resonance imaging (MRI).

In a further aspect, the present invention provides a diagnostic method of medical imaging a subject using a radiotracer compound or radiotracer composition as described herein, the method including the steps of:

(i) administering an effective amount of the radiotracer compound to the subject; and then

(ii) imaging the subject. In particular, the diagnostic method of medical imaging is PET imaging, optionally in combination with computed tomography (CT) or MRI.

The radiotracer compounds and radiotracer compositions described herein are useful in the imaging of cells in a subject that include the sodium/iodide symporter. The radiotracer compounds described herein (having the radiolabeled anion [ ¹⁸ F]S03F ^" , [ ¹⁸ F]PF6 ^" or

[ ¹⁸ F]PC>2F2 ^" ) have an affinity for such cells. Accordingly, the radiotracer compounds and compositions described herein may, in particular, be used in a method of imaging the thyroid gland (e.g. the thyroidal follicular cells), salivary glands, gastric mucosa, thymus, lacrimal glands, kidney and/or lactating mammary glands in a subject.

The diagnostic method of medical imaging may be used for the diagnosis or detection of any disease or disorder associated with a tissue or organ of the subject having NIS-expressing cells. For example, the diagnostic method of imaging may be for the detection or diagnosis of any disease or disorder associated with the thyroid gland (e.g. the thyroidal follicular cells), salivary glands, gastric mucosa, thymus, lacrimal glands, kidney, Meckel's diverticulum, ovary and/or breast (e.g. lactating mammary glands) in a subject.

In an embodiment, the disease or disorder associated with these tissues or organs is a proliferative condition of the thyroid gland, salivary gland, gastric mucosa, thymus, lacrimal gland, kidney, Meckel's diverticulum, ovary or breast (e.g. mammary gland). The term "proliferative condition," as used herein, pertains to an unwanted or uncontrolled cellular proliferation of excessive or abnormal cells which is undesired, such as, neoplastic or hyperplastic growth. In some embodiments, the treatment is treatment of a proliferative condition characterised by benign, pre-malignant, or malignant cellular proliferation, including but not limited to tumours and cancers.

In other embodiments, the disease or disorder is an autoimmune disease or disorder associated with the thyroid gland, salivary glands, gastric mucosa, thymus, lacrimal glands, kidney, Meckel's diverticulum, ovary or breast (e.g. lactating mammary glands). In further embodiments, the disease or disorder is hypothyroidism or hyperthyroidism (such as congenital hyperthyroidism).

In particular, the radiotracer compounds and compositions may be useful in the detection, diagnosis and/or monitoring of thyroid cancer. In some embodiments, the radiotracer compounds and radiotracer compositions described herein may be used in a method of imaging tissues in which NIS expression has been induced by gene therapy methods. Such gene therapy methods include gene therapy of cancer to enable tumours to be treated with 1-131 or other NIS substrates. Alternatively, the gene therapy method may be an experimental model for the study of a cancer.

In addition, the radiotracer compounds and compositions may be used in reporter gene imaging. NIS may be used as a reporter gene in gene therapy and imaging of cell migration and differentiation (Blower et al., 2014). Radiotracer compounds and compositions described herein may be used in a method of medical imaging wherein the radiotracer compound is administered to a subject with cells transfected with the NIS reporter gene. Transfection of cells with the NIS reporter gene is a known process per se.

The radiotracer compounds and radiotracer compositions described herein may be used to track cells transfected with the NIS reporter gene in a method of cellular therapy. In other words, the method of medical imaging may include a step of administering therapeutic cells before administering the radiotracer compound (or radiotracer composition), wherein the at least some of the therapeutic cells are transfected with the NIS reporter gene. The transfected therapeutic cells can be any transfected cell useful in cellular therapy. Examples include, but are not limited to, transfected cells for use in stem cell therapy (such as transfected stem cells), transfected cells for use in transplant therapy (such as transfected transplant cells), and transfected cells for use in cellular immunotherapy (such as transfected CAR T-cells and adoptively transferred T-cells).

The cells transfected with NIS reporter cells may be cells associated with a disease or disorder of the subject. In particular, the transfected cells may be cells associated with one or more of the following cancers: adrenal cancer, anal cancer, bladder cancer, bone cancer, bowel cancer, brain/CNS tumours, breast cancer, cervical cancer, endometrial cancer, esophagus cancer, eye cancer, gallbladder cancer, Hodgkin disease, Kaposi sarcoma, leukemia (such, for example, acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), chromic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML)), liver cancer, lung cancer (such as, for example small cell and non-small cell lung cancer), lymphoma, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, neuroblastoma, non-Hodgkin lymphoma, osteosarcoma, ovarian cancer, pancreatic cancer, pituitary tumours, prostate cancer, retinoblastoma,

rhabdomyosarcoma, sarcoma, skin cancer (such as, for example, basal and squamous cell skin cancer, melanoma, merkel cell skin cancer), stomach cancer, testicular cancer, and uterine cancer.

Dosage

It will be appreciated by one of skill in the art that appropriate dosages of the radiotracer compounds, and radiotracer compositions comprising the radiotracer compounds, can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects. The selected dosage level will depend on a variety of factors including, but not limited to, the radioactivity and specific activity of the particular radiotracer compound described herein, the route of administration, the time of administration, the rate of excretion of the radiotracer compound, the duration of the imaging, other drugs, compounds, and/or materials used in combination, the severity of the condition, and the species, sex, age, weight, condition, general health, and prior medical history of the patient. The amount of radiotracer compound and route of administration will ultimately be at the discretion of the physician, veterinarian, or clinician, although generally the dosage will be selected to achieve local concentrations at the site of action which achieve the desired effect without causing substantial harmful or deleterious side-effects. Administration can be effected in one dose, continuously or intermittently (e.g., in divided doses at appropriate intervals) throughout the course of imaging. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation used for imaging, the purpose of the imaging, the target cell(s) being imaged, and the subject being imaged. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician, veterinarian, or clinician.

In general, a suitable dose of the radiotracer compound or radiotracer composition for a human subject is 100 MBq or more. In some embodiments, the radiotracer compound or radiotracer composition may administered to a human subject in a dose of 200 MBq or more, 300 MBq or more, or 400 MBq or more. In one embodiment, the radiotracer compound or radiotracer composition may administered in a dose of about 500 MBq. The route of administration is not particularly limited. For example, the administration of the radiotracer compound described herein may be parenteral (such as intravenously), enteral (such as orally) or topical (such as epicutaneously). The radiotracer compositions described herein may be administered to a subject parenterally (in other words, by injection). In some embodiments, the radiotracer compositions are administered parenterally as an aqueous solution.

The Subject/Patient

The subject/patient may be any animal or human subject. The subject/patient may be a chordate, a vertebrate, a mammal, a placental mammal, a marsupial (e.g., kangaroo, wombat), a monotreme (e.g. a platypus), a rodent (e.g., a guinea pig, a hamster, a rat, a mouse), murine (e.g., a mouse), a lagomorph (e.g., a rabbit), avian (e.g., a bird), canine (e.g., a dog), feline (e.g., a cat), equine (e.g., a horse), porcine (e.g., a pig), ovine (e.g., a sheep), bovine (e.g., a cow), a primate, simian (e.g., a monkey or ape), a monkey (e.g., marmoset, baboon), an ape (e.g., gorilla, chimpanzee, orangutan, gibbon), or a human. Furthermore, the subject/patient may be any of its forms of development, for example, a foetus. In some embodiments, the subject/patient is a mammalian subject. In further embodiments, the subject/patient is a human subject.

EXAMPLES

General

Abbreviations

cpm - counts per minute

HBSS - Hanks' balanced salt solution

HPLC - high performance liquid chromatography

hNIS - human sodium/iodide symporter

IC - ion chromatography

K222 - Kryptofix 2.2.2

PET - positron emission tomography

PET/CT - positron emission tomography and computed tomography

RCC - radiochemical conversion

RCP - radiochemical purity

RCY - radiochemical yield

R _f - retardation factor

SUV - standardised uptake value

TLC - thin layer chromatography Radioanalytical methods

a) Ion chromatography for F ⁸ F]S03F ^~ :

Column = Shodex IC-I524A (WAX), Flow rate = 1.5 mL/min, Eluent = 2.5 mM phthalic acid + 2.3 mM tris(hydroxymethyl)aminomethane, pH = 5.0; elution time is around 8 minutes.

Alumina plates w/ 100% MeOH running solvent. ¹⁸ F ^" R _f = 0, S0 ₃ ¹⁸ F- R _f = 0.45 .

Example 1 - "" cC uptake inhibition assay of SQ3F ^'

The ICso of S0 ₃ F ^" (unlabelled) for inhibition of ^99m Tc0 ₄ ^" uptake in a human NIS (hNIS) expressing cell line (HCT1 16-C19) was determined experimentally by determining uptake of 99mj _c Q ₄ - j _n ^ _e p _resence of varying concentrations of the SO ₃ F ^" anion. The IC ₅₀ of SO ₃ F ^" was measured at 0.55 μΜ. The IC50 of I ^" using the same procedure was measured at 4.7 μΜ. SO3F ^" therefore displays good potency towards hNIS. Figure 1 shows a graph of uptake of ^99m Tc0 ₄ ^" in a human NIS expressing cell line (HCT1 16-C19) in the presence KSO3F at various concentrations to determine the IC50 value of KSO3F.

Experimental Detail

HCT1 16-hNIS-C19 cells were seeded in 12-well plates at a density of 0.5 x 10 ⁶ cells/well and incubated with 5% CO2 at 37°C for 24 hours prior to experiment. Each well was washed twice with HBSS before incubation with the inhibitory anion (SO3F ^" or I ^" ) in HBSS (700 μΙ_) for 30 minutes. ^99m Tc0 ₄ ^" (0.1 MBq) in HBSS (50 μΙ_) was then added and incubated for a further 30 minutes. The final concentration of blocker in each well ranged between 1 x 10 ^"3 and 1 x 10 ^"13 M. The medium was then removed from each well and washed with HBSS (2 x 750 μΙ_). Cell-bound activity was then extracted with 1 M NaOH (750 μΙ_). Bound and un-bound radioactivity were then measured in a gamma counter and the uptake of the radiotracer expressed as a percentage of the total radioactivity per well. Example 1 b - ^99m TcQ ₄ ^" uptake inhibition assay comparing IC50 of SQ3F ^' and BF ₄ - ^"

The IC50 of S03F- was determined, compared to BF ₄ ^" , in a second cell line, expressing a hNIS/TagRFP fusion protein, MTLn3E.D34-CXCR4-GFP.hNIS-TagRFP (also known as 3E.A-hNIS, Fruhwirth et al., 2014), in the presence of various concentrations of SO3F ^" and BF ₄ ^" . The IC50 values of the two tracers were very similar: 1.57 and 1.53 μΜ respectively (see Figure 1 b).

Experimental Detail

hNIS-expressing cells (MTLn3E.D34-CXCR4-GFP.hNIS-TagRFP, aka 3E.A-hNIS) were plated in 6-well plates at a density of 0.5 ^χ 10 ⁶ cells/well, followed by incubation for 24 hours in a humidified incubator at 37°C with 5% CO2. Medium was then removed and the wells were rinsed twice with 1 mL of Hank's balanced salt solution (HBSS) and then incubated at 37°C with 5% C0 ₂ for 30 min with 50 μί of 0.01 MBq [ ^99m Tc]Tc0 ₄ ^~ in 950 μί of HBSS containing either KSO3F or NaBF ₄ in a concentration range of 10 ^"13 to 10 ^"2 M. The supernatant was then removed and the cells were rinsed with 1 mL of HBSS before the cells were lysed with 1 mL of 1 M NaOH and rinsed with 1 mL of HBSS. Each condition was performed in triplicate.

Example 2 - Synthesis of r ¹⁸ F1SQ ₃ F ^"

A commercially available organic-soluble form of SO3 (sulfur trioxide-pyridine complex) was used as the precursor for reaction with [ ¹⁸ F]KF in the presence of cryptand, Krytpofix 2.2.2 (K222), and potassium carbonate in a water/acetonitrile mixture.

[ ^"e F]KSO _s F

The outcome of the reaction was determined by ion chromatography and TLC (see experimental section for details) which gave a retention time for unreacted ¹⁸ F ^" of -2 minutes, and for [ ¹⁸ F]SC>3F ^" of -8 minutes. Radiochemical conversion (RCC) was determined by integration of the peaks on the radioactivity chromatogram to find the percentage incorporation into the desired product.

Examples 2A-2F

The expected product was formed under all conditions examined:

Entry Precursor (mg) Base Temp. (°C) Time (min) RCC (%)

A 5 KHCOs 80 15 38

B 5 KHCOs 30 15 7

RCC = radiochemical conversion

Example 2E was selected for purification and further use. Purification of the reaction mixture using solid phase extraction methods was carried out by addition of water to the reaction mixture, and passing over an alumina cartridge and QMA cartridge (containing a

silica-based, hydrophilic, strong anion-exchanger with large pore size of 300A, available for example from Waters Ltd, UK as Sep-Pak QMA Plus Light) sequentially. Unreacted fluoride was trapped on the alumina cartridge, while the product passed freely through and was trapped on the QMA cartridge. The QMA cartridge was then washed with H2O to remove chemical impurities and the final product was eluted in 0.9% NaCI or a buffer of choice with sufficient ionic content. The process yields the desired radiotracer with 17% radiochemical yield (RCY), in > 99% radiochemical purity (RCP) and radiochemical identity has been confirmed by co-elution with cold KSO3F using HPLC. In this application, RCY refers to the yield of the isolated product following purification; RCC refers to the percentage of starting solid material in the reaction mixture that has been converted into product.

The pyridine content of the final product was assessed by HPLC and found to be less than 25 g/mL.

Determination of the specific activity of the tracer by analysis of [ ¹⁸ F]S03F ^" content using IC indicated that it was greater than 131.3 GBq/μΓΤΐοΙ. This represents a significant

improvement over the reported specific activity of [ ¹⁸ F]BF4 ^" (1 GBq/μηΊθΙ - see Blower et al., 2010).

Experimental detail

Solutions of K ₂ C0 ₃ (5.2 mg) in H ₂ 0 (0.4 mL) and K222 (14.2 mg) in MeCN (1 .1 mL) were prepared. A portion of the K2CO3 (0.2 mL) was then added to the K222 solution to form the QMA eluent. ¹⁸ F ^" in H2O was passed over a QMA cartridge (preconditioned by passing 10 mL NaHC0 ₃ , 10 mL H ₂ 0), and then eluted with the QMA eluent (0.9 mL). The fluoride was then dried by azeotropic distillation, first at 1 10°C then at 95°C twice further with addition of MeCN (0.4 mL) each time. SO3. pyridine complex (5 mg) in MeCN (1 mL) was then added to the dried residue and heated to 80°C for 10 min. The reaction was then quenched by addition of H2O (2 mL) and passed over a neutral alumina cartridge (preconditioned by passing 20 mL H ₂ 0, 10 mL air) and QMA (preconditioned with 5 mL 1 M NaCI, 10 mL H ₂ 0) cartridge. The QMA was then washed with H2O (4 mL) before the product eluted with 0.9% NaCI (0.4 mL). Example 3a - In Vitro Uptake of r ¹⁸ F1SQ ₃ F-

Uptake of [ ¹⁸ F]S0 ₃ F- in hNIS-expressing cells (HCT1 16-C19), in addition to the parent cell line (HCT1 16, which does not express NIS) in the presence and absence of NaCI0 ₄ (20 μΜ) as a competitive inhibitor was examined (Figure 2a). Significant uptake of the tracer was only observed in the cell line expressing hNIS, in the absence of blocker indicating specific, blockable NIS-mediated uptake. The uptake of the tracer over time in vitro was also examined in HCT1 16-C19 cells (see Figure 2b) and found to begin to plateau around 45 minutes.

Experimental Detail HCT1 16-hNIS-C19 (see Blower et al., Nuclear Medicine

Communications 201 1 , 32:98-105) or HCT1 16 cells were seeded in 6-well plates at a density of 1 x 10 ⁶ cells/well and incubated with 5% CO2 at 37°C for 24 h prior to experiment. Each well was washed twice with HBSS and incubated with or without NaCI0 ₄ (20 μΜ) in HBSS (950 μΙ_) for 30 minutes. 0.1 MBq [ ¹⁸ F]S0 ₃ P in HBSS (50 μΙ_) was then added and incubated for a further 30 minutes. The medium was then removed from each well and the wells washed with HBSS (2 x 1 mL). Cell-bound activity was then extracted with 1 M NaOH (1 mL). Bound and un-bound radioactivity were then measured in a gamma counter and the uptake of the [ ¹⁸ F]S03F ^" radiotracer expressed as a percentage of the total radioactivity per well.

Example 3b - In vitro uptake of [ ¹⁸ F]S0 ₃ F ^" compared to [ ¹⁸ F]BF ₄ ^"

To determine the uptake of [ ¹⁸ F]SOsF ^" in a second cell line, and to compare it with [ ¹⁸ F]BF4 ^" , [ ¹⁸ F]S0 ₃ F- mixed with ^99m Tc0 ₄ ^" was incubated with 3E.A-hNIS cells, and similarly, [ ¹⁸ F]BF ₄ ^" mixed with ^99m Tc0 ₄ ^" was incubated with 3EA-hNIS cells. The uptake data are shown in Figure 2c. In the case of [ ¹⁸ F]SOsF ^" mixed with ^99m Tc0 ₄ ^" , both tracers showed high uptake, but in the case of [ ¹⁸ F]BF ₄ ^" mixed with ^99m Tc0 ₄ ^" , the uptake of each tracer was reduced (see Figure 2c). This shows that the specific activity of [ ¹⁸ F]BF ₄ ^" is sufficiently low that the additional molar concentration required to achieve sufficient radioactivity is enough to saturate the hNIS, causing potential problems in bioassays and PET imaging, whereas [ ¹⁸ F]SC>3F ^" does not suffer from this problem.

Experimental Detail 3E.A-hNIS cells were grown in 6-well plates at a density of 1 ^χ 10 ⁶ cells/well and incubated for 24 h at 37°C with 5% CO2 prior to the initiation of the study. Afterwards, each well was rinsed twice with 1 mL of HBSS and then incubated under the same conditions with 50 μΙ_ of a mixture of 0.1 MBq [ ¹⁸ F]S0 ₃ F- (or [ ¹⁸ F]BF ₄ ^" ) with 0.1 MBq ^99m Tc0 ₄ ^" in 950 μί. of HBSS. Both tracers, [ ¹⁸ F]S0 ₃ F ^" or [ ¹⁸ F]BF ₄ ^" with [ ^99m Tc]Tc0 ₄ ^~ , were added together simultaneously as a mixture to the same well. At time-points of 10 min up to 70-80 min, the supernatant was removed and the cells were rinsed with 1 mL of HBSS. Cell uptake activity was then obtained by lysing the cells with 1 mL of 1 M NaOH with subsequent washing with 1 mL of HBSS. Cell-bound and supernatant radioactivities were then counted in a gamma counter (1282 Compugamma CS Universal Gamma Counter, LKB, Wallac) using simultaneous measurement protocol of F-18 and Tc-99m.

Example 4 - Radiotracer Stability

The stability of [ ¹⁸ F]SOsF ^" in both its final formulation (0.9% NaCI) and in human serum over a 4 hour time period was then examined by ion chromatography as described above, sampling every 1 hour. Ion chromatography was used for the 0.9 % NaCI solutions and radioTLC analysis was used for the serum stability study (TLC method as described above, Rf ¹⁸ F ^" = 0, Rf SC>3 ¹⁸ F ^" = 0.45). No release of ¹⁸ F ^" over this time was observed indicating that the [ ¹⁸ F]SC>3F ^" remained intact and was not subject to degradation/hydrolysis.

Example 5 - In Vivo Behaviour

The in vivo behaviour of the tracer in normal mice was then examined. Female Balb/c mice were injected with around 5 MBq [ ¹⁸ F]SOsF ^" in 100 μί of 0.9% NaCI aqueous solution (saline) via the tail vein. Each animal was then imaged with PET/CT scanning over either:

(i) a 30 minute period commencing at the time of [ ¹⁸ F]SOsF ^" injection (Figure 3a);

(ii) a 30 minute period commencing at the time of [ ¹⁸ F]SOsF ^" injection and with

NaCI0 ₄ administration one hour before SO3F administration) (250 mg/kg) (Figure 3b)). The subjects were then culled and dissected to analyse radiotracer biodistribution. Maximum intensity projections indicated uptake of the tracer at sites of endogenous NIS expression (thyroid, salivary glands, stomach), with rapid clearance from the blood pool and excretion into the urinary bladder. In the presence of perchlorate, selective uptake of the tracer in NIS-expressing organs is no longer observed and instead the blood pool is predominantly visualised. The maximum intensity projection PET/CT images for [ ¹⁸ F]SC>3F ^" (i) at 30 minutes are shown in Figure 3a, and (ii) at 30 minutes with perchlorate block are shown in Figure 3b.

Examination of the ex vivo biodistribution confirms uptake of the tracer in thyroid, stomach and salivary glands which is blocked in the presence of perchlorate. The ex vivo

biodistribution is shown in Figure 4.

Experimental Detail:

Female Balb/c mice under isofluorane anaesthetic were injected with -5 MBq [ ¹⁸ F]SOsF ^" in 100 μΙ_ of 0.9% NaCI via tail vein injection approximately 30 minutes post-injection of either 0.9% NaCI or NaCI0 ₄ (250 mg/kg). PET/CT scans were then acquired for 30 minutes or 2 hours before culling the animal by cervical dislocation. Ex vivo biodistribution was then determined by dissection and organ counting. Uptake in each organ is expressed in terms of a standard uptake value (SUV). This is determined as radioactivity per gram of tissue divided by total radioactivity per gram of whole body mass. Radioactivity and weight of the tail and excreted fluids were not included.

The I Cso of PF ₆ ^" (unlabelled) for inhibition of ^99m Tc0 ₄ ^" uptake in a human NIS (hNIS) expressing cell line (HCT1 16-C19) was determined experimentally by determining uptake of 99mj _c Q ₄ - j _n † _ne p _resence of varying concentrations of the PF ₆ ^" anion. The IC50 of PF ₆ ^" was measured at 0.026 μΜ. The I C50 of I ^" using the same procedure was measured at 4.7 μΜ. PF6 ^" therefore displays good potency towards hNIS.

Experimental Detail

HCT1 16-hNIS-C19 cells were seeded in 12-well plates at a density of 0.5 x 10 ⁶ cells/well and incubated with 5% CO2 at 37°C for 24 hours prior to experiment. Each well was washed twice with HBSS before incubation with the inhibitory anion (PF6 ^" or I ^" ) in HBSS (700 μΙ_) for 30 minutes. ^99m Tc0 ₄ ^" (0.1 MBq) in HBSS (50 μΙ_) was then added and incubated for a further 30 minutes. The final concentration of blocker in each well ranged between 1 x 10 ^"3 and 1 x 10 ^"13 M. The medium was then removed from each well and washed with HBSS (2 x 750 μΙ_). Cell-bound activity was then extracted with 1 M NaOH (750 μΙ_). Bound and un-bound radioactivity were then measured in a gamma counter and the uptake of the radiotracer expressed as a percentage of the total radioactivity per well.

Example 7 - Synthesis of r ¹⁸ F1PF ₆ ^:

Radiolabelling was developed that was carried out in the solid phase using no solvent, in a polypropylene reaction vessel (see schematic below).

(tWWp^p ₍ 1&', 9ρ _] . _Κ ρρ _{6 1 W K0H}

PCI5 » ^» # » l ^,8,19 F]-KPF _e

1 10X, 20 mins [ ^t8 '«FJ.KP0 ₂ F ₂ 100X. 10 mins

To remove free fluoride, the KOH was neutralised with 1 M HCI, before passing over a neutral alumina column. Analysis of radiochemical identity was by reverse-phase ion chromatography using a C4 column on a HPLC apparatus. This gave a retention time of -7 minutes for PF6 ^" and 2-3 minutes for all other anions. Samples were heavily diluted before analysis to reduce their ionic strength. Fractions of eluate were collected and measured in a gamma counter. The distribution of the radioactivity shows that [ ¹⁸ F]PF6 ^" is the sole radioactive species present. This gave a radiochemical yield of 0.1 %, which could be increased to 1.2% by using a tenfold increase of PCI5.

Experimental Details

1 ⁸ F ^" in H2O was passed over a QMA cartridge (preconditioned 5 mL 1 M KCI, 10 mL H2O), and then eluted with a solution of KF (3.5 mg) in H2O (0.5 mL) into a polypropylene reaction vessel. MeCN was added (0.4 mL) and the fluoride was then dried by azeotropic distillation at 100°C, and twice further with addition of MeCN (0.4 mL) each time. PCI ₅ (2 mg) in Et ₂ 0 (1 mL) added as a suspension to the reaction vessel before drying as before. The remaining solids were heated to 1 10°C for 20 minutes, then 1 M KOH (0.5 mL) was added and continued to heat at 105°C for 10 minutes. 1 M HCI (0.5 mL) was added and passed over a neutral alumina cartridge (preconditioned 20 mL H2O, 10 mL air) to obtain the product.

Example 8 - In Vitro Uptake

Uptake of [ ¹⁸ F]PF ₆ ^" in hNIS-expressing cells (HCT1 16-C19), in addition to the parent cell line (HCT1 16, which does not express NIS) in the presence and absence of NaCI0 ₄ (20 μΜ) as a competitive inhibitor was examined (see Figure 5). This demonstrated specific, blockable NIS-mediated uptake. Experimental Detail

HCT1 16-hNIS-C19 or HCT1 16 cells were seeded in 6-well plates at a density of 1 x 10 ⁶ cells/well and incubated with 5% CO2 at 37°C for 24 h prior to experiment. Each well was washed twice with HBSS and incubated with or without NaCI0 ₄ (20 μΜ) in HBSS (950 μί) for 30 min. Radiotracer (5 KBq [ ¹⁸ F]PF ₆ ^" ) in HBSS (50 μί) was then added and incubated for a further 30 minutes. The medium was then removed from each well and washed with HBSS (2 x 1 mL). Cell-bound activity was then extracted with 1 M NaOH (1 mL). Bound and unbound radioactivity were then measured in a gamma counter and the uptake of the radiotracer expressed as a percentage of the total radioactivity per well. Example 9 - Alternative synthesis of f ¹⁸ F1PF6 ^z

Phosphorus pentafluoride-pyridine complex may be used as the precursor for reaction with [ ¹⁸ F]KF in the presence of cryptand, Kryptofix 2.2.2 (K222), and potassium carbonate in a water/acetonitrile mixture.

Purification of the reaction mixture using solid phase extraction methods may be carried out by addition of water to the reaction mixture, and passing over an alumina cartridge and QMA cartridge (containing a silica-based, hydrophilic, strong anion-exchanger with large pore size of 300A, available for example from Waters Ltd, UK as Sep-Pak QMA Plus Light) sequentially. Unreacted fluoride may be trapped on the alumina cartridge, while the product passes freely through and trapped on the QMA cartridge. The QMA cartridge may then be washed with H2O to remove chemical impurities and the final product may be eluted in 0.9% NaCI or a buffer of choice with sufficient ionic content.

Experimental detail

Solutions of K2CO3 (5.2 mg) in H ₂ 0 (0.4 mL) and K222 (14.2 mg) in MeCN (1.1 mL) are prepared. A portion of the K2CO3 (0.2 mL) may then be added to the K222 solution to form the QMA eluent. ¹⁸ F ^" in H2O may be passed over a QMA cartridge (preconditioned by passing 10 mL NaHCOs, 10 mL H ₂ 0), and then may be eluted with the QMA eluent (0.9 mL). The fluoride may then be dried by azeotropic distillation, first at 1 10°C then at 95°C twice further with addition of MeCN (0.4 mL) each time. PF ₅ . pyridine complex (5 mg) in MeCN (1 mL) may then be added to the dried residue and heated to 80°C for 10 min. The reaction may then be quenched by addition of H2O (2 mL) and passed over a neutral alumina cartridge (preconditioned by passing 20 mL H2O, 10 mL air) and QMA (preconditioned with 5 mL 1 M NaCI, 10 mL H2O) cartridge. The QMA may then be washed with H2O (4 mL) before the product may be eluted with 0.9% NaCI (0.4 mL). Example 10 - ^99m Tc04 ^" uptake inhibition assay of PO2F2 ^"

The IC50 of PO2F2 ^" (unlabelled) for inhibition of ^99m Tc0 ₄ ^" uptake in a human NIS (hNIS) expressing cell line (HCT1 16-C19) was determined experimentally by determining uptake of 99mj _c Q ₄ - j _n † _ne p _resence of varying concentrations of the PO2F2 ^" anion. The IC ₅₀ of PO2F2 ^" was measured at 6.9 μΜ. The IC50 of I ^" using the same procedure was measured at 4.7 μΜ. PO2F2 ^" therefore displays good potency towards hNIS.

Experimental Detail

HCT1 16-hNIS-C19 cells were seeded in 12-well plates at a density of 0.5 x 10 ⁶ cells/well and incubated with 5% CO2 at 37°C for 24 hours prior to experiment. Each well was washed twice with HBSS before incubation with the inhibitory anion (PO2F2 ^" or I ^" ) in HBSS (700 μΙ_) for 30 minutes. ^99m Tc0 ₄ ^" (0.1 MBq) in HBSS (50 μΙ_) was then added and incubated for a further 30 minutes. The final concentration of blocker in each well ranged between 1 x 10 ^"3 and 1 x 10 ^"13 M. The medium was then removed from each well and washed with HBSS (2 x 750 μΙ_). Cell-bound activity was then extracted with 1 M NaOH (750 μΙ_). Bound and un-bound radioactivity were then measured in a gamma counter and the uptake of the radiotracer expressed as a percentage of the total radioactivity per well.

Example 11 - Synthesis of r ¹⁸ F1PF ₆ and r ¹⁸ F1P0 ₂ F ₂ ^' mixture

Radiolabelling was developed that was carried out in the solid phase using no solvent, in a polypropylene reaction vessel (see schematic below). r Kr ^:" 'f ]-KPF,;

PC

1 10X ^, 20 nuns f ^{? ?} ' :UKPO _; P _?

To remove free fluoride, the mixture may be passed over a neutral alumina column. Analysis of radiochemical identity may be by reverse-phase ion chromatography using a C4 column on a HPLC apparatus. Samples may be heavily diluted before analysis to reduce their ionic strength. Fractions of eluate may be collected and measured in a gamma counter.

Experimental Details

1 ⁸ F ^" in H2O was passed over a QMA cartridge (containing a silica-based, hydrophilic, strong anion-exchanger with large pore size of 300A, available for example from Waters Ltd, UK as Sep-Pak QMA Plus Light) (preconditioned 5 mL 1 M KCI, 10 mL H ₂ 0), and then eluted with a solution of KF (3.5 mg) in H2O (0.5 mL) into a polypropylene reaction vessel. MeCN was added (0.4 mL) and the fluoride was then dried by azeotropic distillation at 100°C, and twice further with addition of MeCN (0.4 mL) each time. PCI ₅ (2 mg) in Et ₂ 0 (1 mL) added as a suspension to the reaction vessel before drying as before. The remaining solids were heated to 1 10°C for 20 minutes.

The mixture may then be passed over a neutral alumina cartridge (preconditioned 20 mL H2O, 10 mL air) to obtain the product.

Example 12 - Alternative synthesis of f ¹⁸ F1PQ2F2 ^:

Monofluoromonophosphate-pyridine complex may be used as the precursor for reaction with [ ¹⁸ F]KF in the presence of cryptand, Krytpofix 2.2.2 (K222), and potassium carbonate in a water/acetonitrile mixture.

Purification of the reaction mixture using solid phase extraction methods may be carried out by addition of water to the reaction mixture, and passing over an alumina cartridge and QMA cartridge (containing a silica-based, hydrophilic, strong anion-exchanger with large pore size of 300A, available for example from Waters Ltd, UK as Sep-Pak QMA Plus Light) sequentially. Unreacted fluoride may be trapped on the alumina cartridge, while the product passes freely through and trapped on the QMA cartridge. The QMA cartridge may then be washed with H2O to remove chemical impurities and the final product may be eluted in 0 NaCI or a buffer of choice with sufficient ionic content.

Experimental detail

Solutions of K2CO3 (5.2 mg) in H ₂ 0 (0.4 mL) and K222 (14.2 mg) in MeCN (1.1 mL) may be prepared. A portion of the K2CO3 (0.2 mL) may then be added to the K222 solution to form the QMA eluent. ¹⁸ F ^" in H2O may be passed over a QMA cartridge (preconditioned by passing 10 mL NaHCOs, 10 mL H ₂ 0), and then eluted with the QMA eluent (0.9 mL). The fluoride may then be dried by azeotropic distillation, first at 1 10°C then at 95°C twice further with addition of MeCN (0.4 mL) each time. PO2F. pyridine complex (5 mg) in MeCN (1 mL) may then be added to the dried residue and heated to 80°C for 10 minutes. The reaction may then be quenched by addition of H2O (2 mL) and passed over a neutral alumina cartridge

(preconditioned by passing 20 mL H2O, 10 mL air) and QMA (preconditioned with 5 mL 1 M NaCI, 10 mL H2O) cartridge. The QMA may then be washed with H2O (4 mL) before the product is eluted with 0.9% NaCI (0.4 mL).

REFERENCES

1 . Chung et al., Eur. J. Nucl. Med. Mol. Imaging 2010, 37:2105-2107

2. Blower et al., Eur. J. Nucl. Med. Mol. Imaging 2010, 37:2108-21 16

3. Blower et al., Nuclear Medicine Communications 201 1 , 32:98-105

4. Fruhwirth et al. J Nucl Med 2014; 55:686-694

5. Khoshnevisan A, Jauregui-Osoro M, Shaw K, Bagufia Torres J, Young JD,

Ramakrishnan NK, Jackson A, Smith GE, Gee AD, Blower PJ. [18F]tetrafluoroborate as a PET tracer for the sodium/iodide symporter: the importance of specific activity. Eur J Nucl Med Mol Imaging Res 2016; 6:34.